Nucleoside thiophosphoramidites

ABSTRACT

The present invention relates to new and useful nucleoside thiophosphoroamidites and polynucleotide phosphorodithioate compounds as well as the processes whereby these compounds may be used for synthesizing new mononucleotides and polynucleotides having phosphorothioate and phosphorodithioate internucleotide linkages.

The inventions described herein were supported, in part, with federalfunds under a grant or award from the Department of Health, Education,and Welfare. Accordingly, the United States Government has certainstatutory rights to the invention described herein under 35 U.S.C. 200et seq.

This is a divisional application Ser. No. 08/332,829 filed Oct. 31,1994, which is a Continuation-In-Part application of our earlier filedU.S. patent application Ser. No. 08/012,532, filed on Feb. 2, 1993, nowabandoned; which in turn is a divisional application of U.S. patentapplication Ser. No. 643,381, filed Jan. 1, 1991, now U.S. Pat. No.5,218,103; which in turn is a continuation-in-part of U.S. patentapplication Ser. No. 488,805, filed Mar. 5th 1990, now abandoned (whichin turn is a continuation of U.S. patent application Ser. No. 367,645,filed Jun. 19th 1989, now abandoned, which in turn is a continuation ofU.S. patent application Ser. No. 198,886, filed May 26th 1988, nowabandoned) and U.S. patent application Ser. No. 417,387, filed Oct. 5th1989, now abandoned; which in turn is a continuation in part of U.S.patent application Ser. No. 314,011, filed Feb. 22nd 1989, nowabandoned; which in turn is a continuation in part of U.S. patentapplication Ser. No. 198,886 filed May 26th 1988, now abandoned.

This invention described and claimed herein relates to novel and usefulphosphorous compounds which are particularly useful in the production ofpolynucleotides having analogs attached to phosphorous.

The present invention relates to novel and useful nucleosidethiophosphoramidite, polynucleotide dithioate phosphoramidite,polynucleotide thiophosphoramidite, nucleoside3'-hydrogenphosphonodithioates,nucleoside-3'-yl-S-aralkylphosphorodithioate, nucleoside3'-hydrogenphosphonothioate, nucleoside 3'-methylphosphonothioate,dinucleoside H-phosphonothioate, dinucleoside phosphorodithioate andnucleoside 3'-amidophosphorodithioate compounds as well as the processeswhereby these compounds can be used for synthesizing novelmononucleotides and polynucleotides having phosphorodithioate,methylphosphonothioate and H-phosphonothioate internucleotide linkages,and phosphorothioamidate, phosphorothiotriester, and phosphorothioatesubstituents. These novel mononucleotides and polynucleotides can beused for many biological, therapeutic and diagnostic applications.Potential therapeutic applications include treating tumors, viralinfections and bacterial infections. Additionally, these compounds canbe used to deliver to specific sites in cells and tissues such reagentsas metal ions, toxins, intercalating agents and other reagents thatalter the biochemical reactivity of polynucleotides and proteins. Thesecompounds can also be joined to sugars, steroids, proteins, peptides andlipids so as to deliver these oligonucleotides to specific cells andthus to target certain cells for various biological and therapeuticapplications with these oligonucleotide analogs. These compounds canalso be used for various diagnostic purposes. By attaching fluorescentor other chemically reactive reagents, antigens, antibodies, proteins,and metal ions to these compounds, they can be used for diagnosingdiseases and the normal and abnormal biochemistry of cells, tissues andbody fluids such as blood and urine. There are also many uses in modernbiology and chemistry as well. For example, these compounds can be usedto develop improved methods for sequencing and cutting DNA, for imagingin X-ray crystallography, NMR, and electron microscopy, and for studyingenzyme reactions.

High yielding methodologies are currently available for the rapidsynthesis of sequence defined polynucleotides having the naturalinternucleotide linkage (Caruthers, M. H., Science 230, 281-285, 1985;Caruthers, M. H. and Beaucage, S. L., U.S. Pat. No. 4,425,732;Caruthers, M. H. and Matteucci, U.S. Pat. No. 4,458,066). An importantstep in this process is oxidation of the intermediate phosphite triesterto the naturally occurring phosphate triester with aqueous iodine. Thesephosphite triesters can also be oxidized under anhydrous conditions withamines or ammonia and iodine to yield variable reported amounts ofoligonucleotide phosphoramidates or with sulfur to yield oligonucleotidephosphorothioates (Uznanski, B., Koziolkiewicsz, M., Stec, W. J., Zon,G., Shinozuka, K. and Marzili, L., Chemica Scripta 26, 221-224, 1986;Nemer, M. H. and Ogilvie, K. K., Tetrahedron Letters 21, 4149-4152,1980). Other methods employing H-phosphonate internucleotide linkagescan also be used to synthesize oligonucleotide phosphoramidates andoligonucleotide phosphorothioates (Froehler, B. C., Tetrahedron Letters27, 5575-5578, 1986). A process has also been developed for synthesizingmethylphosphonothioate internucleotide linkages (Brill, W. K.-D. andCaruthers, M. H., Tetrahedron Letters 28, 3205-3208, 1987).Unfortunately, none of these procedures can be used to synthesizepolynucleotides containing the phosphorodithioate or thephosphorothioamidate internucleotide linkages.

The production of uridine 2',3'-cyclic phosphorodithioate is describedin the literature (F. Eckstein, J. Am. Chem. Soc. 92, 4718-4732, 1970).Unfortunately, the process cannot be used to synthesize deoxynucleosidephosphorodithioates or nucleoside phosphorodithioates useful forsynthesizing polynucleotides containing the dithioate linkage. Theprocedure also yields a mixture of mononucleotides havingphosphorodithioate and phosphorothioate moieties. Additionally the yieldor uridine 2',3'-cyclic phosphorodithioate is only 28% and the acidityof P₂ S₅ and the high temperatures used in the synthesis of the cyclicphosphorodithioate would preclude the use of this procedure withprotected deoxyadenosine which would undergo depurination.

Similarly, adenosine cyclic 3',5'-phosphorodithioate can be synthesizedby treating suitably protected adenosine with4-nitrophenylphosphoranilidochloridothioate followed by cyclization withpotassium t-butoxide and conversion to the dithioate in a reaction withsodium hydride/carbon disulfide (J. Boraniak and W. Stec, J. Chem. Soc,Trans. I, 1645, 1987). Unfortunately these reaction conditions and thelow synthesis yields preclude the use of this chemistry for synthesizingoligonucleotides having the phosphorodithioate linkages.

In general, the compounds, according to the present invention, can berepresented by general formulae Ia, Ib, and IIa-f. ##STR1## Where,throughout the following description, R₁ is H or a blocking group; A isD or DR₂ wherein D is OH, H, halogen, SH, NH₂ or azide and DR₂ isoxygen, sulfur or nitrogen as D and R₂ is a heteroatom substituted orunsubstituted blocking group; B is a nucleoside or deoxynucleoside base;R₃ is H or a blocking group, and T, G, X and M are substituents whereinheteroatoms are linked covalently to phosphorous. Substituents T, G, Xand M may also be covalently linked to heteroatom substituted orunsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylakyl, alkenyl,cycloalkenyl, alkynyl, aralkynyl or cycloalkynyl groups. The compoundsof general formulae I and II wherein T, G, X and M are substituentswherein heteroatoms are linked to phosphorus include those in which theheteroatoms are sulfur, nitrogen and oxygen.

The novel compounds of general formula I are of two classes, Ia and Ib;class Ia consists of those in which phosphorus is single bonded to eachof two substituents, X and M, through the heteroatoms; and class Ib arethose in which phosphorous is single and double bonded to sulfur andalso to one other substituent through the group T. These compounds areuseful for synthesizing polynucleotides containing phosphorodithioate,phosphorothioamidate, phosphorothioate triesters and phosphorothioateinternucleotide linkages and for various biological uses.

Compounds of general formula II are those in which phosphorus is doublebonded to sulfur or oxygen and single bonded to hydrogen or thesubstituents T, G, X or M. The preferred compounds are those in whichphosphorous is double bonded to sulfur and single bonded to sulfurjoined to either H or R₄ wherein R₄ is a heteroatom substituted orunsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl or cycloalkynylgroup. The substituent M is sulfur single bonded to phosphorous and toR₅ wherein R₅ is a heteroatom substituted or unsubstituted alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl,alkynyl, aralkynyl or cycloalkynyl. The substituents G and X arenitrogen single bonded to phosphorous wherein G is amino or primaryamino, NHR₆, and X is secondary amino NR₆ R₇. R₆ and R₇ when takentogether form an alkylene chain containing up to 5 carbon atoms in theprincipal chain and a total of up to 10 carbon atoms with both terminalvalence bonds of said chain being attached to the nitrogen atom to whichR₆ and R₇ are attached; and wherein R₆ and R₇ taken separately eachrepresent heteroatom substituted or unsubstituted alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl,aralkynyl, or cycloalkynyl groups; and R₆ and R₇ when taken togetherwith the nitrogen atom to which they are attached may also form anitrogen heterocycle including at least one additional heteroatom fromthe group consisting of nitrogen, oxygen or sulfur; and R₆ and R₇ whentaken together with the nitrogen atom to which they are attached mayalso form a ring nitrogen heterocycle compound which containsunsaturated bonds in the ring structure and may also contain at leastone additional heteroatom from the group consisting of nitrogen, oxygenor sulfur. The substituent T is oxygen single bonded to phosphorus andto hydrogen or R₈ wherein R₈ is a heteroatom substitute or unsubstitutedalkyl, aryl, aralakyl, cycloalkyl, cycloalkylalkyl, alkenyl,cycloalkenyl, aralkenyl, alkynyl, aralkynyl, or cycloalkynyl. Compoundsof general formula II may also be those in which oxygen is double bondedto phosphorous plus M which is single bonded to phosphorous. CompoundsII are useful for various biological uses and for synthesizingpolynucleotides containing phosphorodithioate, phophorothioamidate,phosphorothioate triester and phosphorothioate internucleotide linkageswhich are also useful for biological studies.

Amines from which the substituent group G can be derived include a widevariety of primary amines such as methylamine, ethylamine, propylamine,isopropylamine, aniline, cyclohexylamine, benzylamine, polycyclicamines, heteroatom substituted aryl or alkylamines, and similar primaryamines. Amines from which the substituent group X can be derived includea wide variety of secondary amines such as dimethylamine, diethylamine,diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine,methylcyclopropylamine, ethylcylohexylamine, methylbenzylamine,methycyclohexylmethylamine, butylcyclohexylamine, morpholine,thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine,piperazine, and heteroatom substituted alkyl or aryl secondary amines.

The nucleoside and deoxynucleoside bases represented by B in the aboveformulae are well known and include purines, e.g. adenine, hypoxanthine,guanine, and their derivatives, and pyrimidines, e.g. cytosine, uracil,thymine, and their derivatives.

The blocking groups represented by R₁, R₂ and R₃ in the above formulaeinclude trityl, methoxytrityl, dimethoxytrityl, pivalyl, acetyl,tetrahydropyranyl, methoxytetrahydropyranyl, phenoxyacetyl,isobutyloxycarbonyl, t-butyldimethylsilyl, triisopropylsilyl, alkyl oraryl carbonoyl, and similar blocking groups well known in the art.Common blocking groups represented by R₄ and R₅ include 4-chlorobenzyl,2,4-dichlorobenzyl, β-benzoylmercaptylethyl, and β-cyanoethyl. AlthoughR₁₋₈ can represent blocking groups and in many cases these blockinggroups are removed at some point during synthesis, these radicals mayalso remain covalently attached to nucleosides, nucleotides, andpolynucleotides following synthesis and correspond to fluorescentprobes, antigens, steroids, sugars, peptides, proteins, lipids or othergroups that are useful for a large number of therapeutic, diagnostic,biological or biochemical applications.

As used herein the symbols for nucleotides and polynucleotides areaccording to the IUPAC-IUB Commission of Biochemical Nomenclaturerecommendations (Biochemistry 9, 4022, 1970). Several chemical terms asused in this invention are further defined as follows: These definitionsapply unless, in special cases, these terms are defined differently:

alkyl--a non-cyclic branched or unbranched hydrocarbon radical havingfrom 1 to 20 (preferably 1 to 12) carbon atoms. Heteroatoms, preferablyoxygen, sulfur, or nitrogen can replace or be bonded to the carbonatoms, preferably 1 to 4 carbon atoms in this non-cyclic branched orunbranched radical. Certain heteroatoms such as halogens can be bondedto the carbon atoms in this radical.

aryl--an organic radical derived from an aromatic hydrocarbon by theremoval of one hydrogen atom. This radical can contain one or moreheteroatoms, preferably sulfur, nitrogen, or oxygen, as part of thearomatic ring system. Heteroatoms, preferably halogen, sulfur, oxygen,or nitrogen, can also replace hydrogen attached to carbon that is partof the ring system.

aralkyl--an organic radical in which one or more aryl radicals,preferably 1 to 3, are substituted for hydrogen atoms of an alkylradical.

cycloalkyl--a cyclic hydrocarbon radical containing from 3 to 20(preferably 4 to 12) carbons with 4 to 10 carbons being in the cycle andthe remainder attached to the cycle. Heteroatoms, preferably oxygen,sulfur, and nitrogen, can replace or be bonded to the carbon atoms inthis cyclic hydrocarbon radical. Certain heteroatoms such as halogenscan be bonded to the carbon atoms in this cyclic radical.

cycloalkylalkyl--an organic radical in which one or more cycloalkylradicals, preferably 1 to 3, are substituted for hydrogen atoms of analkyl radical containing from 1 to 20 atoms, preferably 1 to 12 carbonatoms.

alkenyl--an aliphatic, unsaturated, branched or unbranched hydrocarbonhaving at least one double bond and 2 to 20 (preferably 3 to 10)carbons. Heteroatoms, preferably sulfur, oxygen, and nitrogen, canreplace saturated carbon atoms in this radical or be bonded to thesaturated carbon atoms. Heteroatoms such as halogens can be bonded tothe saturated carbon atom. Heteroatoms such as oxygen, sulfur, andnitrogen can also replace certain carbons at an unsaturated positionwhich result in the generation of ketone, thioketone, or imine,respectively.

aralkenyl--an organic radical with one or more aryl radicals, preferably1 to 3, are substituted for hydrogen atoms of an alkenyl radical.

cycloalkenyl--a cyclic hydrocarbon radical having from 3 to 20(preferably 4 to 12) carbons, and at least one double bond. the cyclicpart of this radical would be preferable 5 to 10 carbon atoms with theremainder attached to the cycle. Heteroatoms, preferably oxygen, sulfurand nitrogen, can replace saturated carbons in this radical or be bondedto the saturated carbons. Heteroatoms such as halogens can be bonded tothe carbon atoms in this radical.

alkynyl--an aliphatic, unsaturated branched or unbranched hydrocarbonradical containing at least one triple bond and 2 to 20 (preferably 3 to10) carbons. Heteroatoms, preferably oxygen, sulfur, and nitrogen, canreplace or be bonded to saturated carbons in this radical. Heteroatomssuch as nitrogen can be replaced carbon at an unsaturated position togenerate a nitrile.

aralkynyl--an organic radical in which one or more aryl groups,preferably 1 to 3, are substituted for the hydrogen atoms of an alkynylradical.

cycloalkynyl--a cyclic hydrocarbon radical containing from 6 to 20carbon atoms, preferably 7 or 12 carbon atoms, and at least one triplebond in the cycle with the remaining carbon atoms attached to the cycle,Heteroatoms, preferably oxygen, sulfur, and nitrogen, can replacesaturated carbon atoms in this radical. Heteroatoms such as halogens canbe bonded to the saturated carbon atoms.

Heteroatom substituted radicals--In all these radicals, including alkyl,aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, aralkenyl,cycloalkenyl, alkynyl, aralkynyl, and cycloalkynyl, heteroatoms,preferably sulfur, oxygen, nitrogen, and halogens, can replace hydrogenatoms attached to carbons. As described in the definition for eachradical, heteroatoms, preferably oxygen, sulfur and nitrogen, canreplace carbon atoms at saturated positions in alkyl, aralkyl,cycloalkyl, cycloalkylalkyl, alkenyl, arakenyl, cycloalkenyl, alkynyl,aralkynyl, and cycloalkynyl radicals. Heteroatoms, preferably sulfur,oxygen and nitrogen can also replace carbon as part of the aromatic ringsystem in aryl radicals. Heteroatoms can also replace certain carbonatoms as part of unsaturated systems such as wherein oxygen replacescarbon in an alkene to generate a ketone or aldehyde and nitrogenreplaces carbon in an alkyne to generate a nitrile. Examples of commonheteroatoms substituted radicals used in nucleotide chemistry areβ-cyanoethyl, 4-chlorobenzyl, 2,4-dichlorobenzyl, 4-chlorophenyl,2,4-dichlorophenyl, acetyl, tetrahydropyranyl, di-p-methoxytrityl, andbenzoyl radicals.

phosphorodithioate internucleotide linkage--an internucleotide linkagehaving the general formula 5'-nucleoside-O--P(S)S--O-nucleoside-3' or5'-nucleoside-O--P(S)SH--O-nucleoside-3' which can be illustrated withthe following structure wherein B and A are as defined previously:##STR2## phosphorothioate internucleotide linkage--an internucleotidelinkage having the general formula 5'-nucleoside-OP(O)S--O-nucleoside 3'or 5'-nucleoside-OP(OH)S--O-nucleoside 3' which can be illustrated withthe following structure wherein B and A are as defined previously:##STR3## phosphorothioamidate internucleotide linkage--aninternucleotide linkage having the general formula5'-nucleoside-O--PSNHR6-O-nucleoside-3' and 5'-nucleoside-O--P(S)NR₆ R₇-O-nucleoside-3' which can be illustrated with the following structurewherein B and A are as previously defined: ##STR4## S-alkyl orS-arylphosphorothiotriester internucleotide linkage--an internucleotidelinkage having the general formula 5'-nucleoside-O--P(O)SR₅--O-nucleoside-3' which can be illustrated with the following structurewherein B, A, and R₅ are as previously defined: ##STR5## O-alkyl orarylphosphorothiotriester internucleotide linkage--an internucleotidelinkage having the general formula 5'-nucleoside-O--P(S)OR₈--O-nucleoside-3" which can be illustrated with the following structurewherein B, A and R₈ are previously defined: ##STR6## H-phosphonothioateinternucleotide linkage--an internucleotide linkage having the generalformula 5'-nucleoside-O--P(S)H--O-nucleoside-3' which can be illustratedwith the following structure wherein B and A are as previously defined:##STR7##

A more complete understanding of the terms, and scope of the presentinvention may be obtained in reference to the following figures andexamples, all of which are illustrative of the present invention and arenot to be taken as limiting the scope and breadth of the presentinvention in any manner.

In the figures,

FIGS. 1a and 1b depict the stability in human serum of modifiedpolynucleotides containing phosphorodithioate linkages on the 3'- and5'-ends of the polynucleotide as compared with an unmodifiedpolynucleotide;

FIGS. 2 and 3 depict the antisense efficacy of modified polynucleotideshaving phosphorodithioate linkages as compared with unmodifiedpolyphosphodiester control using primary murine spleen cells; and

FIG. 4 depicts the comparative antisense efficacy of modifiedpolynucleotides having phosphorodithioate linkages as compared withunmodified polyphosphodiester control using rat cells.

The general reaction scheme A for synthesizing compounds Ia, VIIa, andVIIb from which the preferred compounds Ia, IIa and IIc are a subset isshown in the following overview: ##STR8##

The preferred reaction scheme A for synthesizing compounds Ia, IIa, andIIc is represented as follows: ##STR9## wherein R₁, R₃, B, A, X, and Mare as previously defined. Compounds VIIa and IIa are those in whichphosphorous is linked through single bonds to nucleosides and to sulfurand through a double bond to sulfur. compounds VIIb and IIc are those inwhich phosphorous is linked through single bonds to nucleosides and tosulfur and through a double bond to oxygen.

The process of reaction scheme A involves condensation of IIIa with IVawhich can be 2,4-dichlorobenzylmercaptyl-bis(diisopropylamino)phosphineor 4-chlorobenzylmercaptyl-bis (diisopropylamino)phosphine to yield Ia.Reaction of Ia with Va and an activator (e.g. 5-substituted tetrazolesand substituted triazoles, alkylammonium salts, aralkylammonium salts,substituted and unsubstituted pyridinium salts of tetrafluoroborate, andsubstituted and unsubstituted pyridinium and imidazolium salts of acids,5-substituted tetrazoles, halogenated carboxylic acids andN-hydroxybenzotriazole) yields VIa, the dinucleoside2,4-dichlorobenzylthiophosphite or dinucleoside4-chlorobenzylthiophosphite, which can be preferably oxidized withsulfur to yield IIa, the dinucleoside phosphorodithioate triester. Ofcourse oxidation with t-butylperoxide yields IIc, the correspondingdinucleoside phosphorothioate triester.

A second reaction scheme B was also discovered for the purpose ofsynthesizing compounds IIa and additionally IIb, IId, IIe, and IIf. Thegeneral reaction scheme B for synthesizing compounds IIa, IIb, IId, IIeand IIf is as follows: ##STR10##

The preferred reaction scheme B is represented as follows: ##STR11##wherein R₁, R₃, B, A, X, M, G and T are as previously defined. CompoundsIIa, IIa-1, IIb, IIb-1, IId, IIe, and IIf are those in which phosphorousis double bonded to sulfur and single bonded to nucleosides and oneother substituent from the group of substituents including hydrogen, X,M, T and G.

The process of reaction scheme B involves synthesis of VIIIa andcondensation with Va to yield IXa. Reaction of IXa with H₂ S and anactivator such as tetrazole yields the dinucleoside H-phosphonothioate,IId, which can be chemically converted by oxidation with sulfur toIIa-1, the dinucleoside phosphorodithioates; by oxidation with iodine inthe presence of amines to IIe or IIf, the phosphorothioamidates; byalkylation of the dinucleoside phosphorodithioate (IIa-1) to IIa, thephosphorodithioate triesters; by oxidation with iodine in the presenceof alcohols to IIb, the phosphorothioate triesters; and by oxidationwith aqueous iodine to IIb-1, the phosphorothioates.

The present novel compounds of general structure II having differentheteroatoms containing substituents covalently linked to phosphorous canthus be prepared by processes A and B. In some cases wherein phosphorousis double bonded to sulfur and single bonded to nucleosides and to M toyield a dinucleoside phosphorodithioate, processes A and B can both beused to prepare the same compound IIa. For certain others such as IIc,wherein phosphorous is double bonded to oxygen and single bonded tonucleosides and to M, only process A can be used to produce thiscompound. Alternatively compounds IIb, IIb-1, IIe, and IIf havingphosphorous double bonded to sulfur and single bonded to nucleosides andto X or G or T can only be synthesized by process B. It can therefore beseen that both processes of the present invention are required in orderto synthesize all the compounds described by IIa-f. Process A alsoillustrates how compound Ia can be used to synthesize polynucleotideshaving phosphorodithioate and S-aryl or S-alkyl phosphorothioatetriesters as internucleotide linkages. Process A when used to synthesizepolynucleotides can be completed either on art form polymer support orin the absence of these supports.

Of course the nucleoside moiety of the present invention can includemore than one nucleoside and may include a number of nucleosidescondensed as having one or more phosphorous moieties (as shown in IIa-f)in combination with additional internucleotide phosphate diesterlinkages. These polynucleotides having a mixture of internucleotidelinkages, and the presently described linkages as in IIa-f, are preparedusing the novel processes comprising one aspect of the present inventionin combination with preferably conventional phosphoramiditemethodologies for synthesizing the other polynucleotide linkages(although other methods such as phosphate triester, phosphate diester,and H-phosphonate procedures can also be used to synthesize theseadditional linkages). These condensation steps are best carried out onpolymer supports although nonpolymer support procedures can also beused.

The present invention is particularly useful in the chemical synthesisof any deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) containingnovel DNA or RNA compounds having analog substituents G, T, X or M plussulfur double bonded to phosphorus at one or more internucleotidephosphorus containing linkages as found in DNA and RNA.

The synthesis of compounds according to the general formula Ib can berepresented by the following general reaction scheme C: ##STR12##

The preferred reaction scheme C is represented as follows: ##STR13##wherein R₁, B, A, and T are as previously described. Compounds Ib arethose in which all compounds have phosphorus double bonded to sulfur andsingle bonded to a nucleoside, sulfur and T.

The process of scheme C involves synthesis of XIII and XIIIa from IIIaand XII or XIIa. Reaction of XIII or XIIIa with H₂ S and an activatorsuch as tetrazole yields a novel compound, XIVa, the nucleosideH-phosphonothioate, which can be chemically converted by oxidation withsulfur to Ib, the nucleoside phosphorodithioates.

The preferred novel compounds according to the present invention arethose compounds of general formula Ia and IIa wherein for Ia, M is asubstituent having sulfur bonded to phosphorus and to R₅ wherein R₅ is aheteroatom substituted or unsubstituted blocking group, A is H, R₁ is adimethoxytrityl group, B is a nucleoside or deoxynucleoside base havingart form blocking groups, and X is a secondary amino group; and for IIa,sulfur is double bonded to phosphorous, M is a substituent having sulfursingle bonded to phosphorus and to R₅ wherein R₅ is a heteroatomsubstituted or unsubstituted blocking group, A is H, R₁ is adimethoxytrityl group, B is a nucleoside or deoxynucleoside base havingart-recognized blocking groups, and R₃ is H. Of course included in thispreferred group of compounds is IIa-1 wherein M is replaced by thesulfhydryl group, SH.

The novel compound IIa-f of the present invention can be prepared asshown in scheme B from art-recognized starting materials such as VIIIa,a nucleoside 3'-phosphorodiamidite. The initial reaction is accomplishedby dissolving the nucleoside in an organic solvent such as dioxane ortetrahydrofuran containing triethylamine to take up the liberatedhydrochloric acid and adding a bis(dialkylamino) chlorophosphine. Theresulting nucleoside phosphorodiamidite is reacted without isolationwith a second nucleoside. The isolated product of this reaction is adinucleoside dialkylamino phosphoramidite which can be reacted withhydrogen sulfide and tetrazole in an organic solvent such asacetonitrile to yield the dinucleoside H-phosphonothioate, IId. Furtherreaction of the isolated dinucleoside H-phosphonothioate with elementarysulfur in an organic solvent such as a mixture of toluene and lutidineyields the dinucleoside phosphorodithioate, IIa-1. Reaction of thedinucleoside phosphorodithioate with alkyl or aryl halide capable ofalkylating thiols yields the sulfur protecting dinucleosidephosphorodithioate triester, IIa. These novel compounds of the presentinvention can then be used to synthesize polynucleotides havingphosphorodithioate moieties at selected phosphorus internucleotidelinkages. This is possible by first removing the R₃ blocking group byconventional methods from preferably IIa and then reacting this withpreferably an art-recognizing phosphorodiamidite which leads to thedinucleotide 3'-phosphoramidite for use as a synthon in preparingpolynucleotides. Compounds IIa (R₃ =H) can also be converted todinucleotide 3'-phosphate, 3'-phosphate diester, or 3'-H-phosphonate andsynthesis of the polynucleotide then proceeds with these compoundseither on silica-based polymer supports using recognized procedures orin reaction solutions free of polymer supports.

As a further embodiment of the invention, the dinucleosidephosphorodithioates, IIa, are preferably synthesized as shown in schemeA by forming the aralkylmercaptyl-bis(dialkylamino)phosphine, IVa, andthereafter condensing this compound with the selected nucleoside usingtetrazole as an activator in order to form a nucleosideS-(aralkyl)dialkylaminophosphoramidite. The nucleosideS-(aralkyl)dialkylaminophosphoramidite, Ia, can then be condensed with asecond nucleoside using an activator in order to form anS-(aralkyl)dinucleoside phosphite, VIa, which after oxidation withelementary sulfur, yields IIa, the dinucleoside phosphorodithioatetriester. This procedure obviates the requirement for dinucleosidephosphorodithioate triesters as synthons for preparing polynucleotidesand is therefore preferred. Thus the nucleosideS-(aralkyl)dialkylaminophosphoramidite and the art-recognized nucleosidephosphoramidites can be used in any desired sequence in concert witheither elementary sulfur or aqueous iodine oxidation procedures,respectively, to yield polynucleotides having a selected combination ofphosphorodithioate and phosphate internucleotide linkages.

The synthesis of aralkylmercaptyl-bis-dialkylamino phosphine is effectedin an organic solvent solution whereby thebis(dialkylamino)-chlorophosphine is first synthesized and then furthercondensed with an aralkylmercaptan. The first step is reactingphosphorus trichloride in an organic solvent such as tetrahydrofuran ordioxane with a five-fold excess of the dialkylamine. The reactionproceeds smoothly at reflux in a dry atmosphere of nitrogen or argon.The solution of the product is separated from the precipitatedhydrochloride salt of the added amine, and can be concentrated underreduced pressure to a solid. If the dialkylamine is at least as large asdiisopropylamine, this solid can be recrystallized from chemically inertsolvents such as pentane, hexane and heptane. Distillation of thebis(dialkylamino)chlorophosphine is also possible, especially for lowermolecular weight compounds. The next step in the synthesis involvesdissolving an aralkylmercaptan in an inert solvent such as ethyl ether,tetrahydrofuran or dioxane; adding an equivalent of sodium hydride inorder to convert the mercaptan to the mercaptide; and finally adding thebis(dialkylamino)chlorophosphine to the reaction mixture. TheS-aralkylmercaptyl-bis(dialkylamino)phosphine is formed quantitativelyover several hours at room temperature. Removal of sodium chloridefollowed by crystallization from solvents such as acetonitrile ordistillation afford the desired product.

Synthesis of internucleotide bonds containing phosphorodithioatelinkages wherein aralkylmercaptyl-bis (dialkylamino)phosphine is usedfor this conversion requires activating agents which are proton donors.Thus, these phosphines are activated by acidic compounds throughprotonation which facilitates the formation of the desiredinternucleotide bonds containing initially a thiophosphite triester. Theinitial activation step involving thearalkylmercaptyl-bis(dialkylamino)phosphine requires acidic species,preferably mildly acidic, and includes tetrazole and 3-nitrotriazole.The resulting nucleoside aralkylmercaptyl-phosphoramidite can usually beactivated with a tetrazole but may be difficult to activate and in somecases may require a more acidic species such as aromatic amine salts ofstrong acids, para-nitrophenyltetrazole, trifluoromethylphenlytetrazoleand trifluoromethyltetrazolide salts.

The mercaptyl moiety as part of the bis(dialkylamino) phosphine can varyconsiderably in structure. The criteria are that it facilitatesactivation of the mercaptyl-bis (dialkylamino) phosphine by acids, andthat it can be easily removed after termination of the polynucleotidesynthesis. Thus, the preferred mercaptans include benzyl and heteroatomsubstituted benzyl moieties, and heteroatom substituted alkylsubstituents such as β-cyanoethyl or β-benzoyl mercaptyl.

The bis(dialkylamino) moieties, as part of thearalkylmercaptyl-bis(dialkylamino) phosphine, are preferablesubstituents that stabilize both the phosphine and the nucleosidearalkylmercaptylphosphoramidite toward storage and synthesis. Thesedialkylamino groups should also preferably facilitate activation of thephosphine during the reactions leading to the formation ofinternucleotide bonds. These criteria are met most easily bysubstituents such as dimethylamino, diethylamino, diisopropylamino,dipropylamino, dibutylamino, dipentylamino, various isomeric alkylgroups, aralkyl groups, and heteroatom substituted cycloalkyl groupssuch as pyrrolidino and piperidino.

When the present novel compounds are used to form polynucleotides, theyare preferably employed in combination with art recognized nucleosidephosphoramidites. Thus, at sites wherein normal phosphate diesterlinkages are inserted into polynucleotides, art recognized proceduressuch as activation with tetrazole, oxidation with aqueous iodine,capping with acetic anhydride if synthesis is on art-recognized polymersupports, and detritylation with acid are used for synthesis. At thesites wherein phosphorodithioate linkages are to be incorporated intopolynucleotides, a nucleoside, aralkylmercaptyl phosphoramidite isactivated with aromatic amine salts, tetrazole, para-nitrophenyltetrazole, trifluoromethylaryl tetrazole or similar reagents, andfollowing coupling to the growing polynucleotide, the thiophosphiteinternucleotide linkage is oxidized, preferably with elementary sulfurto yield the dithioate. Other steps for utilizing the aralkylmercaptylnucleoside phosphoramidite in the polynucleotide synthesis are the sameas with art recognized nucleoside phosphoramidites. Dinucleosidephosphorodithioate triesters can also be used as synthons forpolynucleotide synthesis. These novel compounds are prepared using thepresently described novel processes. After conversion to preferablyprotected dinucleoside phosphorodithioate 3'-phosphoramidites, they canbe activated with tetrazole and used directly as dinucleotide synthonsvia the normal art-recognized polynucleotide synthesis procedure, eitherpreferably on polymer supports or in the solution phase in the absenceof polymer supports.

Of course once the internucleotide bonds of the polynucleotide have beensynthesized, which included both normal linkages and thephosphorodithioate linkages, the product can, if desirable, be freed ofblocking groups. Thus the first step is treatment with preferablytrialkylammonium thiophenolate to remove the aralkyl blocking group fromthe dithioate moiety and, if methyl groups are used to protect normalinternucleotide linkages, the methyl group from these phosphatetriesters. The remaining blocking groups on sugars, bases, orphosphorus, and also the linkage joining the polynucleotide to a supportif the synthesis had been completed in this manner, can then be removedusing art-recognized procedures such as hydrolysis with aqueous ammonia.If blocking groups on sulfur are used that are labile to reagents otherthan thiophenolate (e.g. trichloroethyl or β-cyanoethyl), then thedeprotection protocol should be modified accordingly.

The following examples and procedures depicting the formation of thecompounds according to the present invention are presented in order toprovide a more complete understanding and illustration of the presentinvention.

EXAMPLE I

Preparation of thiophosphoramidites of the formula: ##STR14##represented as Ia wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl);9-(N-6-benzoyladeninyl); or 9-(N-2-isobutyrylguaninyl); and wherein DMTis di-p-anisylphenylmethyl

The synthesis of these compounds begins with the preparation ofp-chlorobenzylmercaptyl-bis(diisopropylamino) phosphine. Phosphorustrichloride (0.5 mole, 68.665 g, 43.6 ml) was dissolved in 300 mlanhydrous tetrahydrofuran (THF). The PCl₃ solution was cooled to -18° C.by a NaCl ice mixture. Diisopropylamine (2.5 mole, 252.983 g, 350.4 ml)was then added slowly via a dropping funnel. At first the reaction wasviolent and had to be carried out under vigorous stirring (mechanicalstirrer) and cooling. After the reaction to the diisopropylaminodichlorophosphine was complete, the reaction mixture was refluxed for 12hours to afford the desired product. After 12 hours the reaction mixturewas cooled to room temperature and the diisopropylammonium chloride wasremoved by filtration through a Schlenk-fritt. After washing the saltswith THF, the clear reaction mixture was refluxed again for 12 hours toafford the desired product as the only phosphorus containing material inthe reaction mixture (31P-NMR delta 132.4 ppm). The newly formeddiisopropylammonium chloride was removed by filtration and washed withanhydrous ether. The filtrate was evaporated under reduced pressure(rotary evaporator) to a yellowish solid which was recrystallized fromhexanes to afford a colorless crystalline solid. This compound was airstable and moisture insensitive. Para-chlorobenzylmercaptan (50 mmol,7.93 g, 6.6 ml) was dissolved in anhydrous ether (300 ml) and an amountof a sodium hydride suspension in oil (50% NAH in oil) equivalent to 50mmol (2.4 g) was added to the mercaptan solution. As the solution wasstirred (magnetic stirrer), hydrogen evolved indicating the formation ofsodium p-chlorobenzylmercaptide. After two hours,bis(diisopropylamino)chlorophosphine (50 mmol, 13.34 g) was added andthe reaction mixture was stirred until gas evolution stopped (4 hours atroom temperature). 31P-NMR of the reaction mixture indicatedquantitative conversion of the chlorophosphine to the desired productwithout any side reactions (31P-NMR delta 91.4). The salt (sodiumchloride) was removed by filtration through a Schlenk fritt and washedwith anhydrous ether (50 ml). The colorless filtrate was evaporated to awhite foam (p-chlorobenzylmercaptyl-bis(diisopropylamino) phosphine)which was dissolved in a minimum amount of hot acetonitrile (100 ml) andrecrystallized from the same solvent to afford a white crystallineproduct.

The 5'-O-di-p-anisylphenylmethyl nucleoside (5 mmol) andp-chlorobenzylmercaptyl-bis(diisopropylamino)phosphine (6 mmol, 2.33 g)were suspended in dry acetonitrile (15 ml). Tetrazole (10 mmol, 0.69 g)was added and the reaction was stirred for 16 hours at room temperature.The initially present solids (phosphine and nucleoside) dissolved duringthe reaction time and a crystalline solid (diisopropylammoniumtetrazolide) precipitates. After 16 hours, the reaction was quenchedwith pyridine (1 ml) and diluted into acid free ethyl-acetate (100 ml).The solution was extracted twice with an aqueous saturated solution ofsodium bicarbonate and once with brine, successively.

The organic layer was dried over sodium sulfate. After removal of thissalt, the solvent was evaporated in vacuo to afford a glass which wasredissolved in a mixture of chloroform, ethylacetate and triethylamine(45:45:10, v/v/v) and chromatographed on silica gel with the samesolvent. Column chromatography fractions containing the desired productwere combined and the solvent evaporated in vacuo. The product wasdissolved in toluene and precipitated into n-pentane. The nucleosidephosphorothioamidite was isolated after drying the precipitate in vacuoover P₂ 0₅ /KOH (3.33 g, 80.1% yield).

31P-NMR delta 161.3 and 159.97 ppm (two diastereomers) with respect toexternal standard of H₃ PO₄ for the thymidine derivative. 1H NMR delta8.0 (N--H), 7.59 and 7.58 (2×d, JHH=1.2 Hz), 7.42-7.19 (m), 6.83 (d,JHH=8.7 Hz), 6.37 (q, H1'), 4.65-4.58 (m, H3'), 2.05-1.83 (m, H6'),3.80-3.61 (m, CH2 of p-chlorobenzyl), 3.78 (s, H6), 3.48-3.29 (m, H5'),2.45-2.24 (m, H2), 1.44 (Ch3-T), 1.17-1.04 (m, CH3 of isopropyl).

EXAMPLE II

Synthesis of dinucleoside phosphorodithioate triesters of the formula:##STR15## represented as IIa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenymethyl

5'-O-di-p-anisylphenymethylthymidine-3'-S-(p-chlorobenzyl)diisopropylaminophosphoramidite (compound Ia) (0.2 mmol, 166.3 mg) and3'-O-acetylthymidine (0.2 mmol, 56,8 mg) were dissolved in anhydrousdimethylformamide (2 ml). p-Nitrophenyltetrazole (1 mmol, 191.2 mg) wasnext added to this solution. After 15 minutes, the reaction to thedinucleoside thiophosphite was quenched with sulfur (1 mmole, 32 mg).The reaction mixture was then diluted with ethylacetate (50 ml) and thesulfur removed by filtration through a cotton plug. After removal of thesolvents in high vacuo, the desired product was dissolved inethylacetate (10 ml) and extracted twice with aqueous saturated solutionof sodium bicarbonate and once with brine, successively. The organiclayer was dried over sodium sulfate. After removal of the salt, theproduct was chromatographed on silica with a mixture of1.1.1-trichloroethane and methanol (92.5:7.5, v/v). The productfractions were combined and the solvent removed in vacuo. Thedinucleoside phosphorodithioate was dissolved in toluene andprecipitated into n-pentane (31P-NMR delta 97.8, 96.2 with respect to85% H₃ PO₄ as an external standard). FAB-mass spectrum, 1047 (M-), 921(-p-chlorobenzyl), 743 (-DMT), 619 (-DMT and p-chlorobenzyl), 519(3'-O-acetylthymidine 5'-O-p-chlorobenzylphosphorodithioate), 395(3'-O-acetylthymidine 5'-O-phosphorodithioate).

The p-chlorobenzyl group was removed from the phosphorodithioatetriester with a mixture of dioxane:triethylamine:thiophenol (2:2:1,v/v/v) within 1.5 hours at room temperature.

EXAMPLE III

Synthesis of dinucleoside H-phosphonothioate of the formula: ##STR16##represented as IId wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenylmethyl

The first step was condensation of 5'-O-dimethoxytritylthymidine withbis(diisopropylamino)chlorophosphine in dioxane containingtriethylamine. The resulting phosphorodiamidite was reacted withoutisolation with 3'-O-acetylthymidine to yield a homogeneous dinucleosideamidite in 62% yield after silica gel chromatography (5% triethylaminein ethylacetate). Synthesis of the dinucleoside H-phosphonothioateproceeded by dissolving the dinucleoside phosphoroamidite (470 mg. 0.5mmol) in acteonitrile (5 ml), bubbling H₂ S through the solution for 1min, adding tetrazole (35 mg, 0.5 mmol in 1 ml acetonitrile). andfinally stirring the sealed reaction flasks for 16 hours. The reactionmixture was concentrated to a gum on a rotary evaporator, redissolved inethylacetate (50 ml) and extracted twice with 2M triethylammoniumbicarbonate (pH 7.4, 20 ml each). After concentrating in vacuo to a gum,the product as dissolved in dichloromethane (5 ml) and isolated byprecipitation into pentane (400 mg 90%). FAB+mass spectrum, 527 (anhydroDMT dt); FAB-mass spectrum, 890 (M-), 623 (DMT dt-3'-PHO2-), 363 (M-527,5'-PHO₂ -dT-3'-OAc); 31P-NMR delta 71.7 and 70.7 (1 JHP=673.8 Hz and676.3 Hz); 1H NMR delta 7.81 and 7.80 (P--H, 1 JHP=671.4 Hz and 676.7Hz), 7.55 and 7.53 (s, H6), 7.37-7.20 (m, aromatic), 6.82 (d, J=8.8 Hz,DMT), 6.49 and 6.26 (m, H1'), 5.49 and 5.25 (m, H3'), 4.35 (m, H4'),4.19 (m, H5'), 4.07 (m, H4'), 3.76 (s, MeO--DMT), 3.42 (m,H5'),2.54-2.32 (m, H2'), 2.08 and 2.07 (2×s, CH3-acetyl) 1.90 (m, CH₃ -T),1.43 (s, CH₃ -T). Rf=0.35 and 0.28 (methanol/dichloromethane, 1:9, v/v).

EXAMPLE IV

Synthesis of a dinucleoside phosphorodithioate of the formula: ##STR17##represented as IIa-1 wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisyphenylmethyl

Dithymidine phosphorodithioate was synthesized by stirring thedinucleoside H-phosphonothioate (104 mg, 0.1 mmol in 1 mldichloromethane, compound IId) with elementary sulfur (1 mmol in 2 mltoluene: 2, 6-lutidine, 19:1, v/v) for 0.5 hours. Purification viasilica gel column chromatography (0-12% methanol in dichloromethane and0.5% triethylamine) afforded 70% isolated yield. FAB+mass spectrum, 303(DMT+); FAB-mass spectrum, 921 (M-), 395 (5'-PSO₂ -dt-3'-OAc); 31P-NMRdelta 112.7; 1H NMR delta 8.12 (s,NH), 7.90 and 7.60 (2×s, H6),7.40-7.24 (m, aromatic), 6.80 (d, JHP=8.8 Hz, DMT), 6.43 (m, H1'),5,46-5.36 (m, H3'), 4.40 (m, H4'), 4.16 (m, H5'), 3.76 (s, MeO--DMT),3.52 (m, H5'). 2.28 (m, H2'). 2.05 (CH₃ -acetyl), 1.97 (CH₃ -T), 1.58(s, CH₃ -T). Rf=0.14 (methanol/dichloromethane, 1:9, v/v).

The dinucleoside phosphorodithioate was deprotected by standardprocedures and isolated in 86% yield after ether extractions (3×),Sephadex™ G10 gel filtration (H2O), and lyophilization as the ammoniumsalt. FAB+mass spectrum, 579 (M); 31P-NMR delta (D2O) 113.3; 1H NMRdelta 7.60 and 7.46 (2×s, H6), 6.11 and 5.99 (m, H1'), 5.17 (m, H3'),4.85 (m, H3'), 4.15 (m, H4'), 4.03 and 3.62 (m, H5'), 2.21 (m, H2'),1.88 (m, CH3-T). Rf=0.25 (methanol/triethylamine/chloroform, 15:1:84,v/v/v). When the dinucleoside phosphorodithioate was phosphorylated withT4-polynucleotide kinase and gamma-32P! ATP, the rate of kination wasapproximately one-half that of unmodified 3'-5' dithymidine phosphateunder identical conditions. Further testing with snake venomphosphodiesterase (Crotalus adamanteus venom, Sigma) indicated that thephosphorodithioate was stable using conditions wherein the naturaldinucleotide was completely hydrolyzed (assayed by reverse phase HPLC).This compound was also observed to be stable to conc. ammonium hydroxideat 55° C. (16 h) as no degradation or isomerization was observed (³¹P-NMR, thin layer chromatography).

EXAMPLE V

Synthesis of a dinucleoside phophorodithioate 3'-phosphoramidite of theformula: ##STR18## represented as XVa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenylmethyl

In order to introduce the phosphorodithioate linkage intooligonucleotides, a protection/deprotection scheme for thephosphorodithioate internucleotide linkage was developed. Thus, thedinucleoside phosphorodithioate, IIa-1, (57 mg, 0.06 mmol) was alkylatedwith alpha,2,4-trichlorotoluene (50 μl, 1 h, 55 C) in acetonitrile toyield the dinucleoside phosphorodithioate triester quantitatively,Further testing revealed that it was completely stable to reagents usedin DNA synthesis (1% trifluoroacetic acid in dichloromethane and iodinein aqueous lutidine/THF) and that the phosphorodithioate triester wasspecifically S-dealkylated by treatment with thiophenolate(thiophenol:triethylamine:dioxane, 1:1:2, v/v/v. t1/2=3 min at roomtemperature). FAB+mass spectrum, 527 (anhydro DMT dt); FAB-massspectrum, 923 (M+1-dichlorobenzyl), 813 (DMT dt-3'-PSOS-dcb), 553(5'-PSOS-dcb-dT-3"OAc); 31P-NMR (CH₃ CN, ext. lock) delta 94.4 and 93.7,1H NMR delta 7.55 and 7.52 (2×s, H6), 7.37-7.23 (m, aromatic) 681 (d,J+4.6 Hz, DMT), 634 and 6.28 (m, H1'), 5.38 and 5.01 (m, H3'), 4.24-4.08(m, CH₂ -benzyl, H5'+H4'), 3.76 (s, MeO--DMT), 3.42 (m, H5'), 2.39 (m,H2'), 2.08 (s, CH₃ -acetyl), 1.89 and 1.87 (2×s, CH₃ -T). 1.43 and 1.42(2×s, CH₃ -T). Rf=0.74 (methanol/triethylamine/chloroform, 15:1:84,v/v/v.

Conversion to a synthon useful for DNA synthesis was a two step process.The dinucleoside phosphorodithioate triester was first deacylated (the3' acetyl group) using 0.15M tert-butylamine in methanol (0° C., 10 h)and purified by silica gel chromatography to yield IIa (R₃ =H). Lessthan 5% cleavage of the internucleotide linkage (31P NMR, TLC) wasobserved. The deacylated compound was then reacted withbis(diisopropylamino)-2-cyanoethoxy phosphine (1.5 eq) in the presenceof tetrazole (1 eq, 1 h at room temperature) to yield the dinucleosidephosphorodithioate triester as the 3'-phosphoramidite in 76% yield.31P-NMR delta 149.4, 149.4, 148.9 and 97.2, 95.7, 95.5. IH NMR delta7.56 (s, H6), 7.33-7.27 (m, aromatic), 6.84 (d, J=8.5 Hz), DMT),6.39-6.29 (m, H1'), 5.44 (m, H3'), 3.79 (s, MeO--DMT), 1.90 (s, CH₃ -T),1.45 (s, CH₃ -T), 1.18 (d, J=6.6 Hz, CH₃ -iPr). Rf=0.29 and 0.17(chloroform:ethylaceate:triethylamine, 45:45:10, v/v/v). The resultingdinucleotide phosphoramidite, XVa, has been used successfully incombination with unmodified mononucleoside phosphoramidites for thesynthesis of a 26-mer DNA fragment containing the phosphorodithioatelinkage between position 8-9 (98.2% coupling efficiency). The synthesiswas completed on silica based polymeric supports and phosphoramiditecoupling methodologies (U.S. Pat. Nos. 4,458,066 and 4,415,732). Theoligodeoxynucleotide had the following sequence in which the onephosphorodithioate linkage is marked x instead of p.

d(TpGpTpGpGpApApTxTpGpTpGpApGpCpGpGpApTpApApCpApApTpT).

EXAMPLE VI

Synthesis of dinucleoside thioamidates, thiotriester, and thioate of theformulae: ##STR19## represented as IIb and IIf wherein B may be1-Thyminyl; 1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); and9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenylmethyl

The dinucleoside H-phosphonothioate was also found to be useful as aversatile synthon for preparing several analogs rapidly (5 min) inquantitative yield (³¹ P-NMR). Thus when oxidized withiodine/n-butylamine, the phosphorothioamidate (IIf) was isolated in 92%yield. FAB-mass spectrum, 961 (M-), 695 (DMT dt-3'-POSNHBU), 434(5'-POSNHBU-dt-3'-OAc); ³¹ P-NMR delta 74.4 and 74.0; 1H NMR delta 8.36and 8.34 (2×s, NH), 7.59 and 7.56 (2×s, H'), 7.44-7.24 (m, aromatic),6.82 (d, J=8.7 Hz, DMT), 6.41 and 6.28 (m, H1'), 5.28 and 5.23 (m, H3'),4.21 and 4.13 (m, H4' (2×)-H5'), 3.77 (s, MeO--DMT), 3.43 (m, H5'), 2.94(m, CH₂ -N), 2.41 (m, H2'), 2.09 and 2.07 (2×s, CH₃ -acetyl), 1.93 and1.88 (2×s, CH₃ -T), 1.42 (s, CH₃ -T), 1.39-1.23 (m, CH2), 0.90 and 0.83(2×t, J=7.2 Hz and 7.1 Hz, CH₃). Rf=0.56 (methanol/dichloromethane, 1:9,v/v).

The dinucleoside H-phosphonothioate was converted quantitatively to aphosphorothioate triester by oxidation with iodine and 9-anthracenylmethanol (10 equivalents) under anhydrous conditions (IIb). FAB+massspectrum, 527 (anhydro DMT dt); FAB-mass spectrum, 906(m-anthracenylmethyl), 639 (DMT dt-3'-PSO₂ -), 379 (5'-PSO₂ -dt-3'-OAc).³¹ P-NMR delta 51.7 and 51.0. Rf=0.41 (methanol/dichloromethane, 1:9,v/v).

Treatment of the dinucleoside H-phosphonothioate with an aqueoussolution of iodine and pyridine using art form conditions gave thedinucleoside phosphorothioate (IIb) in 87% yield. FAB-mass spectrum, 906(M-), 603 (M--DMT), 379 (5'-PSO₂ -dt-3'-OAc). ³¹ P-NMR delta 60.2 and60.0.

EXAMPLE VII

Synthesis of nucleoside 3'-phosphorodithioate of the formulae: ##STR20##represented as Ib wherein B may be 1-Thyminyl; 1-(N-4-benzoylcytosinyl);9-(N-6-benzoyladeninyl); or 9-(N-2-isobutyrylguaninyl); and wherein DMTis di-p-anisylphenylmethyl

3'-O-(Diisopropylamino)-2-cyanoethyphosphino-5'-O-(di-p-methoxytrityl)thymidine (27.7 mg, 0.04 mmol) was prepared by art form methods (M. H.Caruthers and S. L. Beaucage U.S. Pat. No. 4,415,732) and then dissolvedin anhydrous acteonitrile (440 μl). Hydrogen sulfide was bubbled throughfor 1 min and tetrazole (7.0 mg in 200 μl CH₃ CN, 0.1 mmol) was added.After 10 min ³¹ P-NMH spectroscopy showed quantitative conversion to thediastereomers (delta 70.9 and 70.2 ppm, 1 JPH=675 Hz) of the nucleosideH-phosphorodithioate. ³¹ P-NMR (CH₃ CN) delta 114.0 ppm. FAB-massspectrum, 708 (M-), 182 (M--DMT dt+0). 1H NMR (CDCl₃) 7.53 (s, H6),7.35-6.61 (m, aromatic), 6.15 (t, H1' J=6.4 Hz), 5.12 (m, H3'), 4.20 (m,CH₂ O--P), 2.77 (t, CH₂ CN, J=6.2 Hz), 2.56-2.44 (m, H2'), 1.91 (s, CH₃-T).

Protected nucleoside 3'-phosphorodithioate was dissolved in 80% aqueousacetic acid (4 ml) and left for 30 min at room temperature. The reactionmixture was then diluted with water (4 ml) and extracted 3 timed withether (5 ml). The water phase was evaporated to an oil followed by aco-evaporation with water (5 ml). The oil was redissolved in 25% aqueousammonia and incubated at 55° C. for 16 h, The mixture was re-evaporatedand lyophilized with water to yield the nucleoside3'-phosphorodithioate. FAB-mass spectrum, 338 (M-). FAB+mass spectrum,338 (dt-P+SH=S).

EXAMPLE VIII

Synthesis of nucleoside 5'-phosphorodithioate of the formula: ##STR21##represented as Ib wherein B may be 1-Thyminyl; 1-(N-4-benzoylcytosinyl);9-(N-6-benzoyladeninyl); or 9-(N-2-isobutyrylguaninyl); and wherein DMTis di-p-anisylphenylmethyl.

A solution of N₆ -benzoyl-2-3-methoxymethylideneadenosine (413 mg, 1.1mmol) in anhydrous CHCl₃ (5 ml and tetrazole (76 mg, 1.1 mmol, in CH₃ CN(2.2 m.)) was added 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (345 mg, 1.1 mmol) and stirred at room temperaturefor 20 min. Precipitation of diisopropyl ammonium tetrazolide appearedafter 1/2 min. The reaction mixture was diluted with CH₂ CL₂ (50 ml) andextracted with NaHCO₃ (5% w/v, 50 ml), back-extracted with CH₂ CL₂ (25ml), the organic phase dried over Na₂ SO₄, filtered and evaporated todryness in vacuo. ³¹ P-NMR analysis (CH₃ CN) showed delta 147.9 ppm.Crude product (0.71 g) was dissolved in anhydrous CH₃ CN (5 ml) andbubbled with hydrogen sulfide for 1 min. The reaction mixture was sealedand after 10 min a precipitate of diisopropyl ammonium tetrazolideappeared ³¹ P-NMR (CH₃ CN) delta 72.2 and 71.8 ppm, 1 JPH=669 Hz). Thereaction mixture was evaporated to an oil in vacuo, redissolved inethylacetate (50 ml), extracted with TEAB (1M, pH=7.4, 50 ml), andback-extracted with ethylacetate (50 ml). The combined organic phaseswere dried over Na₂ SO₄, filtered, evaporated, and the oil wasredissolved in CH₂ Cl₂ (5 ml.) Excess elementary sulfur (80 mg, 2.5mmol, in 5 ml toluene/2,6-lutidine, 19:1, v/v) was added. Stirring atroom temperature for 1 h gave the phosphorodithioate product. ³¹ P-NMR(CH₃ CN) delta 114.4 and 114.3. Rf (silica)=0.34 in CH₂ Cl₂ (9:1, v/v).

In addition to those compounds described above, a second aspect of thepresent invention provides novel and useful nucleotides andpolynucleotides having other structure modifications at the phosphorusatom and to the process leading to the synthesis of these compounds.More specifically, the invention describes procedures for synthesizingpolynucleotide phosphorodithioate, H-phosphonothioate, phosphorothioateand phosphorothioamidate compounds from nucleoside-3'-ylphosphorodiamidite and nucleoside-3'-yl phosphorothioamidite synthons.These procedures are especially useful for preparing high molecularweight polynucleotides having these modifications or high molecularweight polynucleotides having these modifications in any combination orin combination with natural internucleotide linkages. The inventiontherefore provides procedures for preparing polynucleotidephosphorodithioate, H-phosphonothioate, phosphorothioate,alkylphosphonothioate and phosphorothioamidate compounds fromnucleoside-3'-yl hydrogen phosphonodithioate,nucleoside-3'-yl-S-aralkylphosphorodithioate and nucleoside3'-methylphosphonothioate synthons. These additional novel procedures ofthe invention are especially useful for preparing polynucleotidephosphorodithioate, polynucleotide phosphorothioate, polynucleotidemethylphosphonothioate and polynucleotide phosphorothioamidate compoundseither exclusively or in any combination including combinations withnatural internucleotide linkages wherein large quantities ofpolynucleotides are required for various uses. The polynucleotidephosphorodithioate compounds synthesized with the nucleoside-3'-ylhydrogenphosphonodithioate and nucleoside-3'-yl-S-aralkylphosphorodithioate synthons also appear to have less contamination ofthe phosphorothioate side-product.

In general, the compounds according to this second aspect of the presentinvention may be represented more specifically than previously described(for example, compound XXI is more specific than compounds Ia, Ib andIIa-f described earlier) by the following general formulae XXI to XXIX:##STR22##

The compounds of general formula XXI, XXII, XXIII and XXIV are usefulfor the synthesis of polynucleotides containing phosphorodithioate,phosphorothioamidate, alkyl or aryl phosphonothioate andphosphorothioate internucleotide linkages which are useful for variousbiological applications. These compounds are also useful for variousbiological applications.

In general, one reaction scheme for the synthesis of compounds XXI,XXII, XXV, XXVI, XXVII and XXIX are shown in the following overview:##STR23##

The preferred reaction scheme for synthesizing compounds XXI, XXII, XXV,XXVI, XXVII and XXIX are shown in the following overview: ##STR24##

The process of the generalized reaction scheme involves first thesynthesis of XXIa and the conversion of this novel compound to variousmononucleotides and oligonucleotides having modified chemicalstructures. The synthesis of XXIa proceeds by reacting XXXa withpreferably bis(triazoyl)chlorophosphine, compound XXXIa, followed by atreatment with H₂ S for five minutes. Various other bisoaminophosphinessuch as tetrazoyl, imidazoyl, diisopropylamino, dimethylamino,diethylamino, morpholino, piperidino and pyrrolidono derivatives areadditional examples of amino groups that can be used. After purging withan inert gas to remove H₂ S, compound XXIa can be isolated bypurification and precipitation. Compound XXIa can then be converted vianovel processes to XXIIa. Thus, when compound XXIa is treated with oneequivalent each of water and dicyclohexylcarbodiimide orN-methyl-2-chloropyridinium iodide in pyridine for 30 minutes, thenucleoside 3'-hydrogenphosphonothioate forms in essentially quantitativeyield. Formation of compound XXIXa via a similar reaction was possibleby treatment of compound XXIa with compound XXXIIIa andN-methyl-2-chloropyridinium iodide. After 15 minutes reaction time,compound XXIXa can be isolated by purification and precipitation fromn-pentane. Thus, compound XXIa can be used to prepare dinucleosidehydrogenphosphonothioates. These novel compounds XXIa are not asreactive as the nucleoside diamidites and not as unstable, but reactreadily with unblocked 3'-OH or 5'-OH of nucleosides under normalreaction conditions. The novel nucleoside hydrogenphosphonodithioatesare stable under normal laboratory conditions to hydolysis and airoxidation and may be stored as dry, stable powders. Therefore, the novelcompounds are more easily employed in the process of forminginternucleotide H-phosphonothioate bonds. The resulting compound XXIXacan then be used to form nucleoside phosphorodithioates, dinucleosidephosphorothioamidates, and dinucleoside phosphorothioates.

The novel compound XXIa may be used to form novel mononucleotidephosphorodithioamidates and dinucleoside phosphorodithioates via a noveloxidative process. The synthesis of compound XXVa, a mononucleotidephosphorodithioamidate, proceeds by treating a pyridine solution ofcompound XXIa with 2-aminoanthracene and iodine to yield compound XXVawhich may be isolated after purification by precipitation fromn-pentane. When XXIa and XXXIIIa in pyridine were treated with oneequivalent iodine, the dinucleoside phosphorodithioate, compound XXVIawas the only detectable product. After addition of sodium bisulfite tooxidize any excess iodine and filtration to remove salts, compound XXVIamay be isolated by purification and precipitation from n-pentane. Thus,compound XXIa can be used to prepare XXVIa, the dinucleosidephosphorodithioate. For preparation of dinucleoside phosphorodithioates,compound XXVIa, the condensation of XXIa with XXXIIIa may be monitoredby decolorization of the iodine solution. This is an especiallyattractive feature as the persistence of the light brown color of excessiodine indicates when the coupling is complete. Furthermore, this novelmethod also appears to be relatively free of reactions which lead tosignificant quantities of phosphorothioates as side products. This isbecause contaminating oxygen does not interfere with the activationprocess and elemental sulfur, which is difficult to solubilize and is arather poor oxidant of P(III) compounds, is not part of thesulfurization reactions.

The resulting novel dinucleoside phosphorodithioate can then be reactedwith various alkylating agents to yield XXVIIa, and this compound maythen be incorporated into polynucleotides.

In addition to this first novel process, a second reaction scheme wasalso discovered for the purpose of synthesizing compounds XXXVIII andXXVII, the completely protected dinucleoside phosphorodithioatetriester. This second scheme is as follows: ##STR25##

The preferred reaction scheme is as follows: ##STR26##

Preferably, R₁, R₄, and R₈ may be removable as blocking groups underdifferent chemical conditions so that each can be selectively eliminatedin the presence of the other. One such preferable combination ofconditions would be R₁ removed with acid (as in the case ofdi-p-methoxytrityl), R₈ removed by a base (as in the case ofβ-cyanoethyl), and R₄ removed by thiophenol (as in the case of2,4-dichlorobenzyl). Of course all other "blocking groups" according tothe invention may also be selected so that each can be selectivelyeliminated in the presence of the others. Using these combinations ofblocking groups, XXVII can be extended to form polynucleotides simply byremoving either R₁ or R₃ preferentially followed by the chemistryoutlined in the scheme immediately above.

The process of the scheme above involves the condensation of nucleosidephosphoramidites such as XXXIVa according to the process in U.S. Pat.No. 4,415,732 to yield XXXVIa. Reaction of XXXVIa without isolation withsulfur yields XXXVIIa which can them be converted to XXXVIIIa withtriethylamine under anhydrous conditions. The triethylammonium salts ofXXXVIIIa may then be stored as a solid. Of course, other bases thatpreferentially remove the R₈ protecting group in the presence of R₄ mayalso be used. Reaction of XXXVIIIa with XXXIIIa in the presence oftriisopropylbenzenesulfonyl chloride then yields XXVIIa, the completelyprotected dinucleoside phosphorodithioate. Of course, other activatingagents such as mesitylenesulfonyl chloride and tetrazolide can be usedto synthesize XXVIIa. Compound XXVIIa may then be further extended tosynthesize larger polynucleotides by removing R₁ from XXVIIa with acidand condensing the resulting compound with XXXVIIIa usingtriisopropylbenzenesulfonyl chloride or tetrazolide as a condensingagent to yield a trinucleotide with two phosphorodithioate linkages.Alternatively, XXVIIa may be treated with a base to remove R₃ and thenconverted to the dinucleoside 3'-phosphoramidite analogous to XXXIVa,using the known conditions in U.S. Pat. No. 4,415,732, which cansubsequently be converted as in the scheme immediately above to adinucleoside-3'-yl-S-aralkylphosphorodithioate analogous to XXXVIIIa.This compound may then be condensed with XXVIIa, wherein R₁ has beenremoved with acid, using triisopropylbenzenesulfonyl chloride to yield atetranucleotide having three phosphorodithioate linkages. Thesepolynucleotides may then be further extended in a similar manner to formlonger polynucleotides having phosphorodithioate linkages or by usingnucleoside 3'-phosphate diesters to polynucleotides having bothphosphorodithioate and phosphate internucleotide linkages.

A third novel reaction scheme that can be used for synthesizing novelcompounds XXIV and XXVIII is depicted below: ##STR27##

The preferred reaction scheme is represented as: ##STR28## wherein R₉ isa heteroatom substituted or unsubstituted alkyl, aryl, aralkly,cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl,aralkynyl, or cycloalkynyl group.

The process of this third reaction scheme starts with anucleoside-3-yl-methylphosphonodiisopropylamidite, XXXIXa, which issulfhydrolyzed with H₂ S and tetrazole to yield XXIVa. Of course, otheramino groups as previously defined by X may replace the diisopropylgroup. Compound XXIVa may then be treated with XXXIIIa in the presenceof one equivalent iodine in pyridine to yield XXVIIIa, and the productpurified by column chromatography. The choice of reaction solvent forthe reaction with iodine is critical as essentially no productcorresponding to XXVIIIa forms when the reaction is carried out indichloromethane.

The preferred novel compounds of this aspect of the invention are thosecompounds of general formula XXI, XXIII, XXIV, and XXVII. Novelcompounds XXI may be used to prepare XXIX, the dinucleosideH-phosphonothioates. Compound XXIX may then be converted to preferablydinucleoside phosphorodithioates (XXVI), dinucleosidephosphorothioamidates and dinucleoside phosphorothioates. Compound XXImay also be condensed with an appropriate nucleoside, XXXIII, withiodine to form XXVI, the dinucleoside phosphorodithioate which can beconverted to XXVII via a conventional alkylating agent. Preferredcompound XXIII can react with an appropriate nucleoside, XXXIII, and acondensing agent such as triisopropylsulfonyl chloride, to form XXVII.

EXAMPLE IX

Synthesis of nucleoside 3'-hydrogenphosphonodithioate (formula XXIa) ofthe formula: ##STR29## wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenylmethyl

To a solution of 25 mmol of PCl₃ (2.18 ml, 3.43 g) in 250 ml CH₂ Cl₂containing 250 mmol (25.3 g, 27.5 ml) of N-methylmorpholine was added83.35 mmol (5.75 g) of 1,2,4-triazole. The reaction mixture was stirredat room temperature for 30 minutes and cooled to 0° C. In this processthe reaction mixture became turbid. ³¹ P-NMR of the reaction mixtureindicated complete formation of chloro-bis-triazolylphosphane (48.1ppm). To this solution was added 5 mmol (2.73 g) ofdi-p-methoxytritylthymidine dissolved in 66 ml of dry CH₂ Cl₂. Afterallowing the reaction mixture to come to room temperature in a period of15 minutes, H₂ S gas was passed through it for an additional period of15 minutes. During sulfhydrolysis, the reaction mixture became clear.After removal of excess H₂ S by passing argon gas through the productmixture, the solvents were evaporated in vacuo. The resulting yellowsolid was taken up in CH₂ Cl₂, and the solution extracted twice with 1Mtriethylammonium hydrogencarbonate. To remove the desired product (³¹P-NMR: 87.5 ppm) from the hydrolysis products (³¹ p-NMR: 113.8 ppm, 52.7ppm), the organic layer was subjected to chromatography (CH₂ Cl₂/EtOZa/CH₃ OH/NEt₃, 60:30:5:5, v:v:v:V) after being dried over NaSO₄.The product fractions were pooled and the product precipitated inton-pentane/ether, (9:1, V:V). The desired product was obtained in 56.6%yield (2.1 g).

EXAMPLE X

Synthesis of the dinucleoside phosphorodithioate (formula XXVIa) of theformula: ##STR30## wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenylmethyl.

0.1 mmol (74.19 mg) 5'-di-p-methoxytrityl-3'-hydrogenphosphonodithioatewas dissolved in 0.9 ml of dry pyridine containing 0.12 mmol (28 mg) of3'-acetylthymidine. To this solution was added drop-wise 110 μl of a 1Msolution of I₂ in pyridine. The reaction mixture decolorized instantlywhen the iodine was added. As the coupling was complete, a brown colorpersisted for at least 5 minutes. The ³¹ P-NMR spectrum of the reactionmixture indicated two peaks: one of the desired product at 115.65 ppm,and a side product at 116.7 ppm (10%). After extraction with aqueoussodium bisulfite, which led to the disappearance of the peak caused bythe side product, the mixture was subjected to column chromatographyusing CH₃ CCl₃ /CH₃ OH/NEt₃ (85:14.5:0.5, v:v:v). The product fractionswere combined and evaporated to dryness. Precipitation from CHCl₃ inton-pentane gave a white solid in 57% (63 mg) yield.

EXAMPLE XI

Synthesis of the nucleoside phosphorodithioamidate of the formula:##STR31## represented as XXVa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); wherein R₉ is 2-anthracenyl; and wherein DMTis di-p-anisylphenylmethyl.

0.1 mmol (74.19 mg) of 5'-di-p-methoxytritylthymidine3'-hydrogenphosphonodithioate was dissolved in 0.9 ml of dry pyridinecontaining 0.12 mmol (23 mg) of 2-aminoanthracene. To this solution wasadded drop-wise 110 μl of a 1M solution of I₂ in pyridine. The reactionmixture decolorized instantly when the iodine was added. As the couplingwas complete, a brown color persisted for at least 5 minutes. The ³¹P-NMR spectrum of the reaction mixture indicated two peaks: one of thedesired product at 95.5 ppm, and a side product at 105.4 ppm (10%).After extraction with aqueous sodium bisulfite, the mixture wassubjected to column chromatography using CH₃ CCl₃ /CH₃ OH/NEt₃(85:14.5:0.5, v:v:v). The fluorescent product fractions were combinedand evaporated to dryness. Precipitation from CHCl₃ into n-pentane gavea yellow solid in 47% (44 mg) yield.

EXAMPLE XII

Synthesis of nucleoside phosphorodithioate triester of the formula:##STR32## represented as XXVIIa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); wherein R₄ is 2,4-dichlorobenzyl; wherein R₃is acetyl; and wherein DMT is di-p-anisylphenylmethyl.

In order to introduce the phosphorodithioate linkage intooligonucleotides, a protection/deprotection scheme for thephosphorodithioate internucleotide linkage was developed. Thus, thedinucleoside phosphorodithioate XXVIa of Example X (57 mg, 0.06 mmol)was alkylated with alpha,2,4-trichlorotoluene (50 μl, 1 hr., 55° C.) inacetonitrile to yield the dinucleoside phosphorodithioate triesterquantitatively. Further testing revealed that this was stable toreagents used in DNA synthesis (1% trifluoroacetic acid indichloromethane and iodine in aqueous lutidine/THF), and that thephosphorodithioate triester was specifically S-dealkylated by treatmentwith thiophenolate (thiophenol:triethylamine:dioxane, 1:1:2, v:v:v,t1/2=3 minutes at room temperature).

Conversion to a synthon useful for DNA synthesis was a two-step process.The dinucleoside phosphorodithioate triester was first deacylated(removal of the 3'-acetyl group) using 0.15M t-butylamine in methanol at0° C. for 10 hours, and purified by silica gel chromatography. Less than5% cleavage of the internucleotide linkage was observed. The deacylatedcompound was then reacted with bis(diisopropylamino)-2-cyanoethoxyphosphine (1,5 eq) in the presence of tetrazole (1 eq) for 1 hour atroom temperature to produce the dinucleotide phosphorodithioate triesteras the 3'-phosphoramidite in 76% yield. The resulting dinucleotidephosphoramidite has been used successfully in combination with modifiedmononucleoside phosphoramidites for the synthesis of a 26-mer DNAfragment containing the phosphorodithioate linkages between positions8-9 (98.2% coupling efficiency). The synthesis was completed on silicabased polymeric supports and phosphoramidite using the teachingscontained in U.S. Pat. No. 4,415,732. The resulting oligonucleotide hadthe following sequence in which the one phosphorodithioate linkage ismarked by x instead of p:

d(TpGpTpGpGpApApTxTpGpTpGpApGpCpGpGpApTpApApCpApApTpT)

EXAMPLE XIII

Synthesis of nucleoside hydrogenphosphonothioate of the formula:##STR33## represented as XXIIa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenylmethyl.

0.1 mmol of the compound according to formula XXIa (74.2 mg) was treatedfor 30 minutes with either 0.1 mmol of DCC and 1 mmol (18 μl) of waterin 1 ml of pyridine, or 0.5 mmol of 2-chloro-1-methyl pyridinium iodidein pyridine. In both cases, the partial hydrolysis was complete. Afterevaporation of the reaction mixture to dryness, extraction with aqueous1M triethylammonium hydrogencarbonate, the reaction mixture wassubjected to column chromatography using CH₃ CCl₃ /CH₃ OH (4:1. v:v)containing 0.5% of triethylamine to yield the desired product.

EXAMPLE XIV

Synthesis of dinucleoside hydrogenphosphonothioate of the formula:##STR34## represented as XXIXa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); wherein R₃ is acetyl; and wherein DMT isdi-p-anisylphenylmethyl.

0.1 mmol (74 mg) of 5'-dimethoxytritylthymidine3'-hydrogenphosphonodithioate was dissolved in 1 ml of dry pyridinecontaining 0.1 mmol (28 mg) of 3'-acetylthymidine and 0.15 mmol (38 mg)of N-methyl-2-chloropyridiniumiodide. After 15 minutes, the ³¹ P-NMRspectrum indicated the formation of mainly the desired product (³¹P-NMR: 71.7 and 70.0 ppm), but also5'-di-p-methoxytritylthymidine-3'-hydrogenphosphonothioate (³¹ P-NMR:52.7 and 52.2 ppm) and unreacted starting material (16%). Afterevaporation to dryness and extraction with aqueous sodium bicarbonateand brine, the reaction mixture was subjected to column chromatographyusing CH₃ CCl₃ /CH₃ OH/NEt₃ (90:9.5:.05, v:v:v). The product fractionswere combined and evaporated to dryness. Precipitation from CHCl₃ inton-pentane produced a white solid product in 45% (40 mg) yield.

EXAMPLE XV

Synthesis of nucleoside S-(4-chlorobenzyl)phosphorodithioate of theformula: ##STR35## represented as XXIIIa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); wherein R₄ is 4-chlorobenzyl; and whereinDMT is di-p-anisylphenylmethyl.

To a solution of 372.4 mg (0.5 mmol) of5'-O-di-p-methoxytritylthymid-3'-yl-O-(β-cyanoethyl)phosphordiisopropylamiditein 2.5 ml dry and deoxygenated CH₃ CN was added 0.22 ml (0.262 g, 1.65mmol) of 4-chlorobenzylmercaptan and a solution of 84,8 mg (1.2 mmol) oftetrazole in 2 ml of CH₃ CN. The reaction mixture was stirred at roomtemperature under argon for 40 minutes, at which time a saturatedsolution of sulfur (2.25 ml) in toluene/2,6-lutidine (19/l) was added.The resulting mixture was allowed to continue to stir at roomtemperature for 1 hour. The mixture was then diluted with EtOAc and theorganic layer was washed with 5% aqueous NaHCO₃, water and saturatedNaCl, dried over MgSO₄, filtered, and evaporated. The crude residueobtained was dissolved in a minimum amount of CH₂ Cl₂ and precipitatedinto pentane to give 0.4 g (96% yield) of colorless solid. Furtherpurification by silica gel chromatography using CH₃ CCl₃ /CH₃ OH/NEt₃(97:2:1, v:v:v) resulted in a certain amount of β-cyanoethyl groupcleavage, and thus pure product was not obtainable.

To a solution of 372.4 mg (0.5 mmol) of5'-O-di-p-methoxytritylthymid-3'-yl-O-(β-cyanoethyl)S-4-chlorobenzenylphosphorodithioate in 2 ml of NEt₃ and 2 ml of CH₃ CNwas stirred at room temperature for 5 hours. Solvent was removed byevaporation and the crude residue was purified by silica gelchromatography using CH₂ Cl₂ /CH₃ OH/NEt₃ (95:3:1, v:v:v) to give 0.29gram foamy compound XXIIIa as the triethylammonium salt (85.7% yield).

EXAMPLE XVI

Synthesis of dinucleoside phosphorodithioate of the formula: ##STR36##represented as XXVIIa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); wherein R₄ is 4-chlorobenzyl; wherein R₃ isacetyl; and wherein DMT is di-p-anisylphenylmethyl.

To a solution of 30 mg (34 μmol) of XXIIIa and 11.62 mg (1.2 eq) of3'-O-acetylthymidine in 0.6 ml dry pyridine was added 30.9 mg (3 eq) oftriisopropylbenzenesulfonyl chloride and 21 μl (21.6 mg; 7.7 eq) of1-methylimidazole at room temperature under argon. The progress of thecoupling reaction was monitored by ³¹ P-NMR. After 25 minutes, thecomplete disappearance of the starting XXIIIa (71.7 and 71.2 ppm) andthe formation of the pyrophosphorodithioate intermediate (81.4 and 81.1ppm) and the desired XXVIIa (95.3 and 94.7 ppm) were observed. Thereaction was complete after 2 hours at room temperature and its ³¹ P-NMRshowed 5 peaks at 99.1 ppm (8,3% intermediate, unidentified product),95.0 and 94.4 ppm (89.2% intermediate, desired dimer5'-O-di-p-methoxytritylthymidine-3'-O-(s-4-chlorobenzyl)-3'-O-(5'-O-thymidylyl-3'-O-acetyl)phosphorodithioate(XXVIIa), and 26.9 and 26.7 ppm (2.5% intermediate, undesiredphosphorothioate dimer). The selectivity of the oxygen vs sulfuractivation of XXIIIa in the above coupling reaction is 97.3:2.7.

EXAMPLE XVII

Synthesis of nucleoside methylthiophosphinate of the formula: ##STR37##represented as XXIVa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); and wherein DMT is di-p-anisylphenylmethyl.

0.5 mmol (389 mg) of5'-dimethoxytrityl-N-4-benzoyl-deoxycytidine-3'-methylphosphonodiisopropylamiditewas dissolved in 3 ml of dry acetonitrile. To this solution was added asolution of 2 mmol (140 mg) of tetrazole in acetonitrile. Subsequently,H₂ S was passed through this solution for 5 minutes. The crude reactionmixture displayed two ³¹ P-NMR signals at 70.7 and 70.5 ppm. The productmixture was then diluted with 50 ml of ethylacetate and was extractedwith aqueous sodium bicarbonate and brine. After drying over sodiumsulfate and removal of salt and solvents, the product was taken up intoluene and precipitated into n-pentane. The product was obtained in94.1% yield (335 mg).

EXAMPLE XVIII

Synthesis of the dinucleoside methylphosphonothioate of the formula:##STR38## represented as XXIVa wherein B may be 1-Thyminyl;1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); or9-(N-2-isobutyrylguaninyl); wherein R₃ is acetyl; and wherein DMT isdi-p-anisylphenylmethyl.

0.1 mmol (71.2 mg)5'-dimethoxytrityl-N-4-benzoyl-deoxycytidine-3'-hydrogenmethylthiophosphinatewas dissolved in 9.0 ml of dry pyridine containing 0.1 mmol (28 mg) of3'-acetylthymidine. To this solution was added, drop-wise, 110 μl of a1M solution of I₂ in pyridine. The reaction mixture decolorized within 2minutes. As the coupling was complete, the brown color persisted for atleast 5 minutes. The ³¹ P-NMR spectrum of the reaction mixture indicatedtwo peaks, one of the desired product at 98.06 and 97.18 ppm, andseveral side-products at 87.05 and 86.58 ppm (30%). After extractionwith aqueous sodium bisulfite, the reaction mixture was subjected tocolumn chromatography using CH₃ CCl₃ /CH₃ OH (9:1, v:v). The productfractions were combined and evaporated to dryness. Precipitation fromCHCl₃ into n-pentane followed. The product was obtained as a white solidin 47% (47 mg) yield. If the reaction was carried out in CH₂ Cl₂, almostno formation of dimer was observed by ³¹ P-NMR. Instead, severalproducts giving NMR-signals from 85.3-93.4 ppm were formed.

EXAMPLE XIX

This example demonstrates synthesis of the monomeric thiophosphoramiditenucleoside intermediates used for the synthesis of "end-capped"polynucleotides; i.e., partially modified polynucleotides containingphosphorodithioate linkages on the 3'- and/or 5'-ends used in laterexamples to demonstrate antisense efficacy. An example is given forsynthesis of the thymidine intermediate, with preparation of theadenosine, cytosine, and guanosine intermediates following a parallelsynthesis scheme.

Dimethoxytritylthymidine (20.2 g, 37.2 mmoles) was dried byco-evaporation in dry pyridine (2×150 ml) followed by co-evaporationwith 1:1 anhydrous methylene chloride/toluene (2×150 ml). The resultingsolid was further dried overnight in a desiccator under vacuum.Methylene chloride (500 ml) was added to dissolve the dried material,with the resulting solution being checked for the absence ofdetritylation using a solvent system of TLC in EtOAc/CH₂ CL₂ /Et₃ N45/45/10). The solution was then added via canula to a mixture oftris-pyrrolidyl phosphine (9.5 ml approximately 40.92 mmoles) and 0.5Mtetrazole solution (9.3 ml, 4.65 mmoles), and stirred for 15 minutesafter addition was complete. A further aliquot of phosphine (0.52 ml,2.2 mmoles) and tetrazole solution (9 ml, 4.5 mmoles) was then added tothe reaction mixture, followed by stirring for 10 minutes. At thispoint, TLC (same solvent system) indicated that all the startingmaterial (dimethoxytritylthymidine) had been consumed. An aliquot (0.11ml, 0.75 mmole) of 1-trimethylsilylimidazole was then added to thereaction mixture, followed by stirring for 10 minutes, after whichtetrazole solution (194 ml, 97 mmoles) was added, followed by injectionof 2,4-dichlorobenzyl mercaptan (9.5 ml, 59.2 mmoles). The resultingmixture was shaken vigorously for 80 (±5) seconds before triethylamine(70 ml) was added, and then the entire reaction mixture was partitionedwith saturated aqueous sodium bicarbonate (700 ml). The organic layercontaining the desired nucleoside intermediate was further washed with10% sodium bicarbonate solution (3×700 ml) and brine (700 ml) beforedrying on sodium sulfate. Following filtration of the solution andwashing of the solids with toluene (120 ml), the combined organicsolvents were concentrated to approximately 100 ml and the nucleosideproduct precipitated from heptane (degassed, 1% triethylamine, 4liters). The thioamidite nucleoside was isolated by filtration and driedunder vacuum to a yield 26.72 g (86% by weight; 92% by ³¹ p NMR). Thephosphoramidite nucleoside was again precipitated by dissolution intotoluene (120-150 ml) containing triethylamine (approximately 10%),filtered, and then precipitated into degassed heptane (1%triethylamine), with the further purified product again being collectedby filtration.

Recovery was improved if the reaction mixture was shaken vigorously foronly 80 seconds. It is believed that limiting the shaking to therecommended 75-85 second time frame avoids the formation of undesired byproducts which can otherwise render the monomeric nucleosideintermediate less effective for polynucleotide synthesis. Specifically,if the recommended time frame is exceeded, the desired addition of asingle sulfur moiety and a single nitrogen moiety to each monomericnucleoside may be disrupted by a disproportionating exchange reaction,resulting in the formation of bis-thio and bis-amino by products. Thelatter by product, in particular, can lead to "branching" of the nascentoligonucleotides which may reduce the amount of correctly assembledpolynucleotide and complicate purification of the desired end product.

The following scheme depicts the general processes used in the polymersupported synthesis of unmodified polynucleotides as described in thefollowing Example XX: ##STR39## wherein in this and the followingscheme, DMT, A and B are as previously defined; wherein CE is cyanoethylderived from the phosphoroamidite reagent; and wherein S/S is a solidsupport such as silica or porous glass as is conventional to solidsupport automated processes for oligonucleotide synthesis.

The following scheme depicts the general processes used in the polymersupported synthesis of phosphorodithioate-modified polynucleotides asdescribed in the following Example XX: ##STR40## wherein R is a moietyfrom the thioamidite reagent as described previously and in thefollowing Example XX.

EXAMPLE XX

This example demonstrates the automated, polymer-supported synthesis ofmodified polynucleotides having one or more terminal phosphorodithioateinternucleotide linkages for use as antisense compounds. Antisensepolynucleotides are designed to be complementary to a specific portionof a target gene, or to the RNA transcribed from a target gene, so thatthe antisense polynucleotide can hybridize to the target gene (or itstranscribed RNA message), thus inhibiting proliferation of the geneticmessage of the target gene and thereby also inhibiting gene expression.The antisense polynucleotide must be effective at physiologicaltemperatures, and is typically from about 12 to about 30 nucleotides inlength. Generally, longer antisense polynucleotides within this rangeare desirable, because they have a lower probability of occurring bychance in large genomes. For example, a 17-mer polynucleotide should beunique to a mammalian genome. On the other hand, if an antisensepolynucleotide is too long (i.e., substantially longer than about 25-30nucleotides), it may hybridize nonspecifically to longer non-targetsequences. This type of nonspecific hybridization is unavoidable,because the physiological body temperature of a patient cannot beadjusted to increase stringency. Ionic antisense polynucleotides, suchas the phosphorodithioate modified polynucleotides of the presentinvention, have a slightly lower t_(m) than nonionic polynucleotides(e.g., naturally occurring phosphodiester-linked polynucleotides) andcan, therefore, be adjusted toward the upper range of 25 to 30nucleotides. The amount of the antisense polynucleotide used to inhibitthe expression of a target gene will depend upon the particular genetargeted by the antisense polynucleotide and other circumstancessurrounding its administration, such as, for example, the tolerancelevel of a patient to whom an antisense polynucleotide is beingadministered. Any amount of antisense polynucleotide which inhibitsproliferation of the genetic message of a target gene is referred toherein as a "proliferation inhibiting amount" of an antisensepolynucleotide.

The modified polynucleotides in this example were synthesized using thethioamidite intermediates from Example XIX on a Model 380A, Model 380B,or Model 394 DNA synthesizer obtained from Applied Biosystems, Inc.(Foster City, Calif.). The synthesis of phosphorodithioate-modifiedpolynucleotides can also be accomplished using other automated or manualsystems.

All synthetic reagents used in this example, with the exception of thesulfurizing reagent (5% elemental sulfur in carbondisulfide/pyridine/triethylamine (23:23:4, v/v/v), carbondisulfide/pyridine (1:1, v/v), anhydrous tetrahydrofuran, andconcentrated ammonium hydroxide, were purchased from either AppliedBiosystems or Glen Research (Sterling, Va.). The sulfur, carbondisulfide, pyridine and tetrahydrofuran reagents were purchased fromAldrich Chemical Co. (Milwaukee, Wis.). Concentrated ammonium hydroxidewas purchased from Fisher Scientific (Pittsburgh Pa.). Tetrahydrofuranwas rendered anhydrous by distillation from metallic sodium andbenzophenone.

Nuclear Magnetic Resonance spectra were recorded on either a Varian VXR®300 megahertz spectrometer (Palo Alto Calif.) or a General ElectricOmega® 300 spectrometer (Fremont Calif.).

The current synthetic cycle for phosphodiester polynucleotide synthesison a solid support consists of four synthetic steps: 1) removal of thedimethoxytrityl group from the 5'-hydroxyl via acid catalysis; 2)coupling the polymer supported nucleoside with a 5'-dimethoxytritylnucleoside-3'-aminophosphine; 3) capping the unreacted polymer supportednucleoside with acetic anhydride and N-methylimidizole; and, 4)oxidizing the phosphite triester to the phosphate triester with aqueousiodine. See also, Beaucage and Caruthers, Tetrahedron Letters, 22,1859-1862 (1981 ). Between the various synthetic steps, washing stepsare also required in order to remove the previous solvents or reagentsfrom the polymer-supported nucleoside/polynucleotide.

A different synthetic cycle was used in this example to synthesizemodified polynucleotides containing one or more phosphorodithioatemodifications. Use of this different cycle enabled insertion of thedesired modification at select locations within a designated modifiedpolynucleotide. The cycle used in this example are referred to asMXS203. The two synthetic schemes executed during the cycle are asdepicted above.

The major synthetic differences between the cycle used to generate themodified polynucleotides containing the phosphorodithioate linkage(s)and normal phosphodiester linkages in this example are: 1) the couplingreagent; and, 2) the oxidation reagents, although numerous differencesin the actual cycle commands were necessary to accommodate the differentcoupling and oxidation steps necessary to generate the desired modifiedlinkages. Typically, in synthesizing a diester (unmodified) linkage, adialkylamino-β-cyanoethylnucleoside phosphine is used as the nucleosideintermediate, with N,N-diisopropylaminophosphines, being used as thenucleoside monomers. In contrast, dialkylaminothionucleoside phosphineswere used to synthesize phosphorodithioate linkages. Typically theN-pyrrolodino-2,4,-dichlorobenzylthionucleoside phosphines, are used.The condensation time for the dialkylaminomethylnucleoside phosphineswas increased due to the slower kinetics of coupling for this couplingreagent. After the coupling step was completed, the resultingthiophosphitodiester was treated with sulfurizing reagent (5% elementalsulfur in carbon disulfide/pyridine/triethylamine (23:23:4, v/v/v), oncefor 75 seconds. Carbon disulfide/pyridine (1:1, v/v) was then used toremove excess sulfurizing reagent.

The following modified polynucleotide (Seq. No. 1) was synthesizedwherein "*" indicates a phosphorodithioate internucleotide linkagebetween the two adjacent nucleosides:

    5'-G*G*C CTG GGA GGT C*C*C* C*A*T-3'

This modified polynucleotide was cleaved from the support, and the baseand phosphate protecting groups removed by treating thepolymer-supported polynucleotide with concentrated ammonium hydroxideovernight at 55° C. The supernatant was decanted and the solventsremoved by evaporation in vacuo. The residue was dissolved in water andre-evaporated to a gum. The resulting gum was dissolved in water andchromatographed on a Sephadex® G50/50 column (Pharmacia, Uppsala,Sweden). The appropriate fractions were pooled and concentrated invacuo.

In order to verify the presence of the phosphorodithioate linkages,concentrated, pooled fractions were consolidated in a single sample thatwas then dissolved in deuterium oxide and evaporated to a powder invacuo. The sample was again dissolved in deuterium oxide, with aphosphorus-31 nuclear magnetic resonance spectrum of the dissolvedsample being taken on a Varian VXR® 300 spectrometer. Resonances at 112ppm and -1 ppm were observed. These resonances correspond to thephosphorodithioate (modified) and phosphodiester (unmodified) moietiesin the partially modified polynucleotide. The relative integration ofthe two signals was 0.79 to 1, correlating with the 7 phosphorodithioate(modified) linkages and the 10 phosphodiester (unmodified) linkagescontained within the modified polynucleotide.

EXAMPLE XXI

This example demonstrates the preparation of phosphorodithioate-modifiedand control (unmodified) polynucleotides for comparison in nucleasedegradation assays. The modified polynucleotides were "end-capped" todetermine an optimal minimum number of phosphorodithioateinternucleotide linkages able to impart nuclease resistance to thepolynucleotide.

Two polynucleotides (Seq. No. 2), each containing the same nucleic acidsequence, were synthesized in this experiment for use in comparing theresistance of the various polynucleotides to nuclease degradation. Themodified polynucleotide contained 5 phosphorodithioate linkages at its5'-end. A corresponding unmodified polynucleotide was also synthesizedfor use as a control. The following nucleotide sequences were generated:

    5'-GGT GGC AGG TCC AGC CAT-3'

    5'-GGT GGC AGG TCC A*G*C* C*A*T-3'

The two polynucleotides used in this example were enzymaticallyphosphorylated according to published procedures by T4 kinase obtainedfrom New England Biolabs (Beverly, Mass.) using γ³² P-adenosinetriphosphate (Maxam and Gilbert, Proc. Nat'l. Acad. Sci., U.S.A., 74,560-564 (1977)), and then subjected to polyacrylamide gelelectrophoresis. The polyacrylamide gel was scanned with aPhosphorimager™ (Molecular Dynamics, Sunnyvale Calif.).

In order to assess the stability of the modified polynucleotides againstnuclease degradation, the kinased polynucleotides were ligatedseparately to some of the following synthetic polynucleotides (Seq. No.3):

    5'-CTG TCA ACA AGT CAC-3'

    5'-C*T*G TCA ACA AGT CAC-3'

    5'-C*T*G* T*C*A ACA AGT CAC-3'.

The ligation reactions were performed according to the publishedprocedure of Yansura et al, Biochemistry, 16, 1772-1776 (1977), using T4DNA ligase obtained from New England Biolabs (Beverly Mass.). Thetemplate (Seq. No. 4) used in the ligation reaction was:

    5'-TGC CAC CGT GAC TTG-3'.

The ligation product was isolated from a 15% denaturing polyacrylamidegel according to Yansura et al, ibid. During the ligation process, theradioactive phosphate atom became internalized within the ligationproduct. This eliminated loss of the label due to phosphatase activityendogenous to the nuclease sources.

EXAMPLE XXII

This example demonstrates the exonuclease resistance of modifiedpolynucleotides having one or more phosphorodithioate internucleotidelinkages on the 3'- and/or 5'-end of the polynucleotide.

Two assays were used to assess the stability of the variouspolynucleotides from Example XX. The first assay used human serum as thenuclease source, with the second assay using HeLa cell cytoplasmicextract as the nuclease source.

Ten milliliters of whole blood were drawn from a human specimen bystandard phlebotomy technique into a red top clot tube purchased fromBecton-Dickenson (Orangeburg N.Y.). The blood was allowed to clot atroom temperature for 30 minutes. The sample was centrifuged at 1000 rpmfor 10 minutes. The serum was removed and 0.5 ml aliquot were prepared.Serum aliquots were stored at -70° C. Aliqouts were thawed immediatelyprior to use.

HeLa S3 cells (American Type Culture Collection, Rockville, Md.) weregrown in suspension in DMEM (Cellgro, Washington D.C.), 10% BFS (Flow),and 1% L-Glutamine (Gibco, Gaithersburg Md.) at a density of 2-5×10⁵cells/ml. Six liters of cells were pelleted for 5 minutes at 1000 rpm,slow cooled to deter lysis and stored at -70 ° C.

Cell cytoplasmic extract was obtained as follows. Thawed cell mass waswashed with 5 PCVs (packed cell volumes) of ice cold PBS and pelleted.Ceils were brought up in 5 PCVs of buffer A (0.1M HEPES/pH 7.5, 1.5 mMMgCl₂, 0.75 mg/ml KCl, 0.5 mM DTT, 0.5 mM PMSF (Sigma Chemical Company,St. Louis Miss.), 1 mg/ml aprotinin (Sigma Chemical Company), 0.5 mg/mlleupeptin (Sigma), 0.7 μg/ml pepstatin (Sigma Chemical Company) to swellcells, left on ice 10 minutes and pelleted. Cells were brought up in 2PCVs Buffer A, doused with an all glass homogenizer (B pestle) andchecked microscopically for lysis. The resulting suspension wascentrifuged for 20 minutes at 25,000 times gravity at 4° C. Thesupernatant (i.e., the cytoplasmic extract) was decanted. Glycerol wasadded to the supernatant to a final concentration of 10% (v/v). Thealiquoted extracts were stored at -70 ° C.

4.5 picomoles of polynucleotide substrate was concentrated to dryness invacuo and dissolved in 7.5 μl of water. 22.5 μl of either serum or HeLacell cytoplasmic extract was added to initiate the assay. The finalconcentration of the polynucleotide was 15μM in 75% (v/v) either serumor cytoplasmic extract. At specific times, 5 μl samples were dilutedwith 5 μl of formamide. 2 μl of 0.1% bromophenol blue (w/v)/0.1% xylenecyanole (w/v) in 80% (v/v) formamide was added to each sample. Thesamples were boiled for two minutes, then placed on ice for two minutes,and then applied to a denaturing 15% polyacrylamide gel. Afterelectrophoresis, the gels were imaged and the amount of full lengthpolynucleotide was quantitated using a Phosphorimager™. All assays wereconducted at least in triplicate. Standard deviations were calculatedusing N in the denominator. Bar graphs of the assay results are depictedin FIGS. 1a and 1b.

In the presence of human serum, "end-capping" at only the 5'-terminus orthe 3'-terminus did not significantly increase the stability of themodified polynucleotide. Specifically, the modified polynucleotides withfive phosphorodithioate linkages at the 3'-terminus, twophosphorodithioate linkages at the 5'-terminus, or fivephosphorodithioate linkages at the 5'-terminus were degraded verysimilarly to the unmodified (phosphodiester) control. However, when boththe 5'- and 3'-ends of the polynucleotide contained phosphorodithioatelinkages, these modified polynucleotides were stabilized relative to theunmodified control. In the case of a modified polynucleotide having fivephosphorodithioate linkages at the 3'-terminus and fivephosphorodithioate linkages at the 5'-terminus, 12-fold more full lengthmodified polynucleotide was present at 24 hours relative to theunmodified control (60% vs. 5%).

In the HeLa cell extract assay system, the two modified polynucleotidescontaining phosphorodithioate linkages at only the 5'-end did notexhibit significant resistance to the cytoplasmic nucleases. However,the modified polynucleotide having five phosphorodithioate linkages atthe 3'-terminus was substantially more stable. At 4 hours, only 5% ofthe unmodified (phosphodiester) control was still intact, while 38% ofthe modified polynucleotide having the five phosphorodithioate linkageson the 3'-end persisted. When both termini contained phosphorodithioatelinkages, 51% of the intact modified polynucleotide was present after 24hours, as compared to less than 2% of the unmodified control.

EXAMPLE XXIII

This example demonstrates the antisense efficacy in murine spleen cellsof selected modified polynucleotides having seven phosphorodithioatelinkages (two phosphorodithioate linkages on the 5'-end and fivephosphorodithioate linkages on the 3'-end).

Four polynucleotides having two phosphorodithioate linkages on the5'-end and five phosphorodithioate linkages on the 3'-end weresynthesized as described in Example XX. The modified polynucleotidesused in this example were given arbitrary numerical designations, asshown below. The symbol "*" indicates the location of phosphorodithioateinternucleotide linkages, and the designation "A" or "C" indicateswhether the partially modified polynucleotide was intended as anantisense or control compound, respectively.

    ______________________________________                                        Designa-                                                                      tion No.                                                                             Sequence                A/C    Seq.                                    ______________________________________                                        E16-19 G*A*G AAC GCT GGA CCT* T*C*C* A*T                                                                     A      5                                       E16-20 A*C*A GGA CTC TCG GCG* C*T*A* C*T                                                                     C      6                                       E16-21 T*C*C ATG TCG GTC CTG* A*T*G* C*T                                                                     A      7                                       E16-22 G*T*A GCT CTC TCG GAT* G*C*C* T*T                                                                     C      8                                       ______________________________________                                    

As a control, partially modified polynucleotides with thephosphorodithioate linkages at the same positions and same sequencescorresponding to E16-19, designated E11-16, and an arbitrary controlsequence, designated E11-10, were prepared. The final controlpolynucleotide was an unmodified phosphodiester polynucleotide havingthe same base sequence as E16-19, designated 397.

For determining the antisense efficacy of the partially modifiedpolynucleotides in this example, murine messenger RNA coding forenvelope C protein was selected as the target because envelope C proteinis believed to inhibit the activity of protein kinase C. Thus, becauseprotein kinase C activity is required for cellular proliferation,inhibition of the expression of envelope C protein should result inmeasurable cellular proliferation. The gene coding for rat envelope Cprotein has a different sequence in the targeted region. As a negativecontrol rat spleen cells were assayed as well.

The assay for determining antisense efficacy was carried out with spleencells as follows. Murine or rat spleen cells (5×10 exp 100 ml well) werecultured in RPMI media supplemented with 10% heat inactivated Fetal CalfSerum (Biofluids), 2 mM I-glutamine, 60 mM β-mercaptoethanol, 100 mg/mlstreptomycin, 100 mg/ml penicillin and 10 mM HEPES in U-bottom 96 wellplates (Costar, Cambridge, Mass.) with or without various concentrationsof the partially modified polynucleotide for 12 to 48 hours in a 37° C.,5% carbon dioxide air incubator. Assays of the modified polynucleotideswere tested in this manner side by side in triplicate. One μCi oftritiated uridine per well was then added. After an additional 6 hours,the cells were harvested. The level of tritium incorporation intocellular RNA was determined, as a measure of cellular proliferation, bybinding cellular RNA to filters and then counting the filters in ascintillation counter.

In the first assay only the four partially modified polynucleotideshaving phosphorodithioate linkages and the unmodified phosphodiestercontrol were tested using primary murine spleen cells. The cells weretreated for 12 hours with three doses of the modified polynucleotide:0.5 μM, 3.3 μM and 30 μM. The phosphodiester compound, sequence 397, wasonly tested at 30 μM. A bar graph of the results is depicted in FIG. 2.The two partially modified phosphorodithioate antisense compounds werevery effective at the lowest dose tested (i.e. 0.5 μM), resulting instimulation indices of 11 and 22 as compared to the control(phosphodiester) polynucleotide at a dose of 30 μM, which resulted in astimulation index of only 7. Based on this experiment, the modifiedpolynucleotides having phosphorodithioate linkages are more than 60-foldmore effective than the unmodified polynucleotides. The controlphosphorodithioate-modified polynucleotides did result in somenon-sequence dependent stimulation, but this was only observed at thehighest dose tested.

In the second assay, the four phosphorodithioate-containingpolynucleotides and the two phosphoromonothioate-containingpolynucleotides were tested. The doses of polynucleotide were 33 nM, 150nM, 660 nM, 2.5 μM and 10 μM and the polynucleotides were administeredfor 24 hours. A graph of the data obtained is depicted in FIG. 3. Thethree control polynucleotides, E16-20, E16-22 and E11-10, did notsignificantly stimulate cellular growth, while the three antisensecompounds did elicit stimulation in a dose dependent manner. The twophosphorodithioate-containing polynucleotides were effective instimulating growth at concentrations as low as 150 nM.

In the third assay only the phosphorodithioate-containingpolynucleotides were tested using rat cells. All four modifiedpolynucleotides were administered at 1.67 μM (i.e., a dose wherein thephosphorodithioate-containing antisense compounds would have beenexpected to elicit a stimulation index of approximately 20, whereas thecontrol phosphorodithioate-containing polynucleotides would have beenexpected to have a negligible effect). All four polynucleotides elicitedapproximately the same effect in rat cells. A bar graph of the data isdepicted in FIG. 8. This finding supports the hypothesis that thesecompounds are acting as sequence specific antisense agents in the murinespleen cells, however, due to sequence divergence between the mouse andthe rat, they are not acting as antisense agents in the rat spleen cellassay.

A listing of all nucleotide sequences described in Examples XX, XXI, andXXIII is provided below:

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCCTGGGAGGTCCCCAT18                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GGTGGCAGGTCCAGCCAT18                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTGTCAACAAGTCAC15                                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TGCCACCGTGACTTG15                                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAGAACGCTGGACCTTCCAT20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ACAGGACTCTCGGCGCTACT20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCCATGTCGGTCCTGATGCT20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTAGCTCTCTCGGATGCCTT20                                                        __________________________________________________________________________

Thus while we have illustrated and described the preferred embodiment ofour invention, it is to be understood that this invention is capable ofvariation and modification and we therefore do not wish to be limited tothe precise terms set forth, but desire to avail ourselves of suchchanges and alterations which may be made for adapting the invention tovarious usages and conditions. Accordingly, such changes and alterationsare properly intended to be within the full range of equivalents, andtherefore within the purview of the following claims.

Having thus described our invention and the manner and process of makingand using it in such full, clear, concise, and exact terms so as toenable any person skilled in the art to which it pertains, or with it ismost nearly connected, to make and use the same,

We claim:
 1. A compound according to the formula: ##STR41## wherein B isa nucleoside or deoxynucleoside base; wherein A is OH, H, halogen, SH,NH₂, azide, or KR₂ wherein K is oxygen, sulfur, or nitrogen, and R₂ is ablocking group; andwherein R₁ is a blocking group.
 2. A compoundaccording to claim 1 wherein the structure of said formula is ##STR42##3. A compound according to claim 1 wherein the structure is ##STR43## 4.A compound according to the formula: ##STR44## wherein B is a nucleosideor deoxynucleoside base; wherein A is OH, H, halogen, SH, NH₂, azide, orKR₂ wherein K is oxygen, sulfur, or nitrogen and R₂ is a blockinggroup;wherein R₁ is a blocking group; and wherein R₄ is a blockinggroup.
 5. A compound according to claim 4 wherein the structure is##STR45##
 6. A compound according to claim 4 wherein the structure is##STR46##
 7. A compound according to the formula: ##STR47## wherein B isa nucleoside or deoxynucleoside base; wherein A is OH, H, halogen, SH,NH₂, azide, or KR₂ wherein K is oxygen, sulfur, or nitrogen and R₂ is ablocking group;wherein R₁ is a blocking group; and wherein R₈ is aheteroatom substituted or unsubstituted alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl,aralkynyl, or cycloalkynyl.
 8. A compound according to claim 7 whereinthe structure is ##STR48##
 9. A compound according to claim 7 whereinthe structure is ##STR49##
 10. A compound according to the formula:##STR50## wherein B is a nucleoside or deoxynucleoside base; wherein Ais OH, H, halogen, SH, NH₂, azide, or KR₂ wherein K is oxygen, sulfur,or nitrogen and R₂ is a blocking group;wherein R₁ is a blocking group;and wherein R₉ is a heteroatom substituted or unsubstituted alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl,alkynyl, aralkynyl, or cycloalkynyl group.
 11. A compound according toclaim 10 wherein the structure is ##STR51##
 12. A compound according toclaim 10 wherein the structure is ##STR52##
 13. A compound according tothe formula: ##STR53## wherein B is a nucleoside or deoxynucleosidebase; wherein A is OH, H, halogen, SH, NH₂, azide, or DR₂ wherein D isoxygen, sulfur or nitrogen, and R₂ is a heteroatom blockinggroup;wherein R₁ is a blocking group; X is an amino group of the formulaNR₆ R₇ (a) wherein R₆ and R₇ each taken separately is a heteroatomsubstituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl orcycloalkynyl, (b) when taken together with the nitrogen atom to whichthey are attached is a heterocyclic containing up to 5 carbon atoms inthe cyclic structure which structure may contain up to an additional 5carbon atoms pendant thereon, (c) when taken together with the nitrogenatom to which they are attached is a nitrogen heterocycle including atleast one additional heteroatom selected from the group consisting ofnitrogen, oxygen and sulfur; and wherein R₅ is a heteroatom substitutedor unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl, or cycloalkynyl.14. A compound according to claim 13 wherein the structure is ##STR54##15. A compound according to claim 13 wherein the structure is ##STR55##16. A compound according to the formula: ##STR56## wherein B is anucleoside or deoxynucleoside base; wherein A is D or DR₂ where D is OH,H, halogen, SH, NH₂ or azide, and DR₂ is oxygen, sulfur or nitrogen as Dand R₂ is a heteroatom substituted or unsubstituted blockinggroup;wherein R₁ is a blocking group; wherein X is a secondary aminogroup of the formula NR₆ R₇,(a) wherein R₆ and R₇ each taken separatelyis a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl,aralkynyl or cycloalkynyl, (b) when taken together with the nitrogenatom to which they are attached is a heterocyclic containing up to 5carbon atoms in the cyclic structure which structure may contain up toan additional 5 carbon atoms pendant thereon, (c) when taken togetherwith the nitrogen atom to which they are attached is a nitrogenheterocycle including at least one additional heteroatom selected fromthe group consisting of nitrogen, oxygen and sulfur; wherein M issulfur; and wherein R₅ is a heteroatom substituted or unsubstitutedalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl,cycloalkenyl, aralkenyl, alkynyl, aralkynyl or cycloalkynyl.
 17. Acompound according to claim 13 wherein R1 is selected from the groupconsisting of trityl, di-p-anisylphenylmethyl, andp-anisyldiphenylmethyl groups.
 18. A compound according to claim 13wherein M is sulfur single bonded to phosphorus and to R₅ wherein R₅ isbenzyl.
 19. A compound according to claim 13 wherein M is sulfur singlebonded to phosphorus and to R₅ where R₅ is a substituent substitutedbenzyl.
 20. A compound according to claim 19 wherein R₅ is selected fromthe group of p-chlorobenzyl, o,p-dichlorobenzyl, a heteroatomsubstituted lower alkyl, and β-cyanoethyl.
 21. A compound according toclaim 13 wherein X is the amino group, NR₆ R₇, where R₆ and R₇ are loweralkyl.
 22. A compound according to claim 21 wherein X is selected fromthe group of diisopropylamino, dimethylamino, diethylamino anddibutylamino.
 23. A compound according to claim 13 wherein B is from thegroup adenine, guanine, cytosine uracil, and thymine.
 24. A compoundaccording to claim 13 wherein X is selected from dimethylamino,diethylamino, diisopropylamino, dibutylamino, methylpropylamino,methylhexylamino, methylcyclohexylamino, ethylcyclopropylamino,methylbenzylamino, methylphenylamino, ethylchloroethylamino,methyltoluyamino, methyl-p-chlorophenylamino,methylcyclohexylmethylamino, bromobutylcyclohexylamino,methyl-p-cyanophenylamino, ethyl-β-cyanoethylamino, morpholino,thiomorpholino, pyrrolidino, piperidino, 2,6-dimethylpiperidino andpiperazino.
 25. A compound according to claim 1 wherein B is selectedfrom the group consisting of 1-Thyminyl, 1-(N-4-benzoylcytosinyl),9-(N-benzoyladeninyl), and 9-(N-2-isobutyrylguaninyl); and wherein R isdi-p-anisylphenylmethyl.
 26. A compound according to claim 4 wherein Bis selected from the group consisting of 1-Thyminyl,1-(N-4-benzoylcytosinyl), 9-(N-benzoyladeninyl), and9-(N-2-isobutyrylguaninyl); and wherein R₁ is di-p-anisylphenylmethyl.27. A compound according to claim 7 wherein B is selected from the groupconsisting of 1-Thyminyl, 1-(N-4-benzoylcytosinyl),9-(N-benzoyladeninyl), and 9-(N-2-isobutyrylguaninyl); and wherein R₁ isdi-p-anisylphenylmethyl.
 28. A compound according to claim 10 wherein Bis selected from the group consisting of 1-Thyminyl,1-(N-4-benzoylcytosinyl), 9-(N-benzoyladeninyl), and9-(N-2-isobutyrylguaninyl); and wherein R₁ is di-p-anisylphenylmethyl.29. A compound according to claim 13 wherein B is selected from thegroup consisting of 1-Thyminyl, 1-(N-4-benzoylcytosinyl),9-(N-benzoyladeninyl), and 9-(N-2-isobutyrylguaninyl); and wherein R₁ isdi-p-anisylphenylmethyl.